CA2222793A1 - Optimized minizymes and miniribozymes and uses thereof - Google Patents

Abstract

This invention is directed to improved catalytic compounds, minizymes and miniribozymes, capable of hybridizing with a target RNA to be cleaved. The minizymes and miniribozymes and compositions of the present invention may be used in vitro or in vivo. They may be used as diagnostic or therapeutic agents.

Description

W O ~/4C9C~ CA 02222793 1997-12-01 PCT/AU96/00343 OPTTMT7.Rn MINIZYMES AND MINIRIBOZYMES AND USES T~BREOF

Throughout this application various references are cited in bracket by author and publication year. The full-citations are listed alphabetically and may be found immediately preceding the claims. These publications are hereby incorporated by reference into the present application.

Bac~ou~d of the Invention Se~eral types of ribozymes have ~een identified in living organisms. One of the first ribozymes to show catalytic turnover was the RNA moiety of ribonuclease P. Ribonuclease P (RNase P) cleaves precursor tRNAs (pre-tRNAs) at their 5' ends to give the mature 5'-termini of tRNAs. In Escherichia coli and Bacillus subtilis, the RNase P holoenzyme is composed of one basic protein subunit of approximate Mr 14,000 (119 amino acids) and one single stranded RNA
molecule of 377 and 401 nucleotides, respectively [Baer, 1990; Altman 1987; Waugh, 1989; Pace, 1990; Nichols, 1988].
Another early ribozyme to show cleavage was the L-19 intervening sequence (IVS) from tetrahymena. The 413 nucleotide intervening sequence (IVS) in the nuclear rRNA
precursor from Tetrah~mena thermoPhila can be excised and the two exons ligated in the complete absence o~ any protein [Kruger, 1982; Cech, 1981~. Unique to this class of self-splicing reaction is the requirement of a guanosine or 5~
guanosine nucleotide cofactor. The h~mm~rhead, which in nature undergoes a self-cleavage reaction, constitutes a third class of ribozymes. A number of plant pathogenic RNAs [Symons, 1989; Symons, 1990; Bruening, 1989; Bruening 1990], one animal ~iral RNA tTaylor, 1990~ and a transcript from satellite II of DNA of the newt tEpstein, 1987; Epstein W096/40906 CA 02222793 l997-l2-ol PCT/AU96/00343 1989] and from a Neuros~ora DNA plasmid ~Saville, 1990]
undergo a site specific self-cleavage reaction in vitro to produce cleavage fragments with a 2',3~-cyclic phosphate and a 5'-hydroxyl group. This reaction is unlike RNase P RNA
cleavage of pre-tRNAs, where the internucleotide bond undergoes a phosphoryl transfer reaction in the presence of Mg" or other divalent cations. Metal cations may be essential to RNA catalysis [Pyle, 1993]. Other reactions documented to date show that ribozymes can catalyze the cleavage o~ DNA [Robertson, 1990; Herschlag 1990], the replication o~ RNA strands [Green, 1992], the opening o~ 2'-3'-cyclic phosphate rings [Pan, 1992], as well as react with phosphate monoesters [Zaug, 1986] and carbon centers [Noller, 1992; Piccirilli, 1992]. Finally, ribozymes with new kinds o~ catalytic reactivity are being created through techniques of in vitro selection and evolution [Breaker and Joyce, 1994; Szostak, 1992].

The ability to design a ribozyme to specifically target and cleave any designated RNA sequence has led to much interest in the potential application of h~mm~rhead ribozymes in transgenic plants and in animal health as gene therapy agents or drugs. To i~ ove the ability to treat a disease or target a specific nucleic acid, it is desirable to optimize the ribozyme to achieve the m~;mllm cleavage activity. While much success has been achieved in vitro in targeting and cleaving a number of designated RNA sequences (Saxena and Ackerman, 1990; Lamb and Hay, 1990; Evans, et al., 1992; Mazzolini, et al., 1992; Homann, et al., 1993), there are ~ewer whole cell examples.

Previous reports have demonstrated that high levels o~
ribozyme expression are required to achieve reduced accumulation of target sequence in vlvo [Cameron and Jennings, 1989; Cotten and Birnsteil, 1989; Sioud and Drilca, 1991; L'Huillier, et al., 1992; Perriman et al., 1993]. Another article suggests a necessity for the target and ribozyme to be sequestered in the same cellular compartment [Sullenger and Cech, 1993]. These reports demonstrate that h~mmerhead ribozymes are clearly capable of specific cleavage of a designated target RNA within a biological system.

SlLmm ar~Y of the In~rention This invention is directed to improved catalytic compounds, minizymes and miniribozymes, capable of hybridizing with a target RNA to be cleaved. The minizymes and miniribozymes and compositions of the present invention may be used in vitro or in vivo. They may be used as diagnostic or therapeutic agents.

W O ~6/4~9~ PCT/AU96/00343 Brief Description of the Fi ~ res Figure 1 shows the CAT Expression (Mean + SEM (Standard Error of the Mean)) for Different treatments. Each ~ experiment is performed in triplicate.

Figure 2 shows the extent of Alamar Blue Reduction (mean +
SEM) in the same experiments as in Figure 1. Extent of reduction relates to metabolic activity of the cells during the 18 hours post-transfection.
Figure 3 shows the cleavage of the IL-2 substrate at 37~C by minizymes (with cctt and gtttt linkers) and a ribozyme with DNA arms.

Figure 4 shows the numbering scheme of (Hertel 1992) for a h~mm~rhead ribozyme and its substrate (Seq ID No. 1-2).

Figure 5 shows time taken for 50~ of an 809-nucleotide interleukin-2 transcript to be cleaved by the miniribozyme IL2MGU W UC (filled circles) and the ribozyme IL2RA (open circles) as a function of temperature.

Sequence of miniribozyme IL2MGU W UC is 5' CAAUGCAA CUGAUGA
~uuuUC GA~AC AGGa 3' (SEQ. ID NO. 49) and ribozyme I~2RA is 5' CAAUGCAA CUGAUGA GUC~UUuu~GAC GAAAC AGGa 3' (SEQ. ID N0.
50)-The target sequence within the IL2 transcript is 5'UCCUGUC*W GCAW G 3' (SEQ. ID NO. 51), where * represents the cleavage site which is 82 nucleotides from the 5' end.
Conditions for cleavage experiment: 50 mM Tris.HCl, pH 8, 10 mM MgCl2, O.2 ~M transcript, 1 ~M miniribozyme or ribozyme, transcript internally labelled, no heat-denaturing or heat-annealing step was performed.

W O'~6/lJ~ CA 02222793 1997-12-01 PCT/AUS'/~

Detailed Descri~tion o~ the Invention This invention is directed to a compound having the ~ormula (Seq ID No. 3):

5 3' (X)n1-A (X) n~ ~5 ' T/U /( X ) a GA
~ ( T/U) b G--X~

wherein each X represents a nucleotide which may be the same or di~erent and Q represents guanidine and may be substituted or modi~ied in its sugar, base or phosphate;
15 wherein each o~ (X) n and (X) n~ represents an oligonucleotide having a predetermined sequence which is capable o~
hybridizing with an RNA target sequence to be cleaved;
wherein each o~ n and n' represents an integer which de~ines the number o~ nucleotides in the oligonucleotide; wherein 2 0 each solid line represents a chemical linkage providing covalent bonds between the nucleotides located on either side thereo~; wherein "a" represents an integer which de~ines a number o~ nucleotides with the proviso that "a" may be O
or 1 and i~ 0, the A located 5' o~ (X) a is bonded to the G
25 located 3' o~ (X) a; and wherein (T/U) b represents an oligonucleotide with the proviso that "b" represents an integer which is 3 or 4.

Alternatively, the compound may have the ~ormula (Seq ID No.
4):

3'(X) n C ~
A U
C- (X) a\ G
~(T/U) ~ G X/
In the compounds above, the oligonucleotide 3'-(X) n~ is 3'-(X)n1-A- or is 3'-(X)n2-C-A-. Pre~erably, (X)a is absent.

W096/40906 CA 02222793 l997-l2-o1 PCT/AU96/00343 The integer "b" of (T/U) b iS preferably equal to 3 or ~.
Preferably, (T/U) b iS a (T) b.

~ The invention is also directed to compositions comprising the compounds above in association with an acceptable carrier; the carrier is preferably a pharmaceutically acceptable carrier.

The invention is also directed to an oligonucleotide transfer vector containing a nucleotide sequence which on transcription gives rise to the compound above. The transfer vector may be a bacterial plasmid, a bacteriophage DNA, a cosmid, an eukaryotic viral DNA, a plant DNA virus, a composite geminivirus, a binary plant expression vector (Ri or Ti) an infective phage particle or a portion thereof.
The packaged oligonucleotide transfer vector may contain promoter sequences for RNA polymerase II, human tRNAVa~, plant tRNA, human tRNA, snRNA promoter or RNA polymerase III. The invention also includes a host cell transformed by the transfer vector. The host cell is a prokaryotic host cell, an eukaryotic host cell, an E. coli host cell, a monkey COS host cell, a Chinese hamster ovary host cell, a mammalian host cell, a plant host cell, a plant protoplast host cell, a hematopoietic host cell, a stem cell, a hematopoietic progenitor cell, a lymphoid cell, T-cell, a B-cell, pre-B cell, a CD4+T-cell or a peripheral blood mononuclear cell.

The invention also provides a method of cleaving a target mRNA in a subject which comprises administering to the subject an effective amount of the compound above or a vector capable of expressing the compound. The administration may be topical in an amount between 1 ng and mg. The administration may also be systemic and administered in an amount between 1 ng and S00 ~g/kg weight/day. The administration may also be aerosol administration. The invention also provides a method of WO~6/1~3C6 CA 02222793 l997-l2-ol PCT/AU96/00343 cleaving a target mRNA in a host cell which comprises administering to the host cell an e~ective amount o~ the compound above.

The compound above may ~urther comprise an antisense nucleic acld which is capable o~ hybridizing with an RNA
target sequence. The compound above may ~urther comprise at least one additional non-naturally occurring oligonucleotide compound which comprises nucleotides whose sequence de~ines a conserved catalytic region and nucleotides whose sequence is capable o~ hybridizing with a predetermined target sequence. The additional non-naturally occurring oligonucleotide compound may be a h~mm~rhead ribozyme, a minizyme, a hairpin ribozyme, a hepatitis delta ribozyme, an RNAase P ribozyme or a combination thereo~. See ~or example h~mm~head ribozyme Haselo~ et al. U.S. Patent No.
5,254,678, issued Oct. 18, 1993; Jennings U.S. Patent No.
5,298,612, issued Mar. 29, 1994; Group I introns, Cech et al. U.S. Patent No. 4,740,463, issued April 26, 1988; Altman et al. U.S. Patent No. 5,168,053, issued Dec. l, 1992 or PCT
International Publication No WO 92/03566), hepatitis delta ribozymes (e.g. Blumenfeld et al. PCT International Application No. WO/90/05157) and hairpin ribozymes (European Patent Application No. EP 360,257, Hampel et al. Nuc. Acids Res. (l990) 18:299-304).

Pre~erred cleavage sites in the target RNA have the sequence "XUX", pre~erably GUC, G W, GUA, W A and W C. By way o~
example, suitable reaction conditions may comprise a temperature ~rom about 4 degree(s) C. to about 60 degree(s) C., pre~erably ~rom about 10 degree(s) to 45 degree(s) C., more pre~erably ~rom about 20 degree(s) to 43 degree(s) C., pH ~rom about 6.0 to about 9.0 and concentration o~ divalent cation (such as Mg2+) ~rom about 1 to about 100 mM
(pre~erably 1 to 20 mM). The nucleotides o~ the sequences (X) n and (X)n, o~ the compounds above may be o~ any number and sequence su~icient to enable hybridization with the nucleotides in the target RNA, as described herein.
Ribozymes containing a small number of nucleotides in each of the groups (X) n and ~X) n~ of the compounds above (such as four nucleotides) would generally be incubated at lower temperatures, such as about 20 degree(s) C. to about 25 degree(s) C. to aid hybridizing with the nucleotide sequences in the substrate. The number of nucleotides n and n' in (X~ n and (X) n' are not necessarily equal. The invention is also directed to covalently-linked multiple ribozymes, where each ribozyme is directed to a target sequence which may be the same or different. In addition these compounds may be covalently attached to an antisense molecule which may be 10 to 100 bases in length. Antisense sequences capable of hybridizing to an RNA in a m~mm~ 1 or plant are well known see (Shewmaker et al. U.S. Patent No.
5,107,065, issued April 21, 1992). As the ribozyme acts as an enzyme, showing turnover, the ratio of ribozyme to substrate may vary widely.

A target RNA cont~;n'ng a suitable cleavage site such as XUX
site may be incubated with a compound described above. The nucleotide sequences (X) n and (X)nl o~ the compounds above are selected to hybridize with their substrate. They may be selected so as to be complementary to nucleotide sequences flanking the cleavage site in the target RNA. On incubation o~ the ribozyme or ribozyme composition and its substrate, an enzyme/substrate complex is formed as a result of base pairing between corresponding nucleotides in the ribozyme and the substrate. Nucleotide sequences complementary to (X) n and (X) n' of the compounds above ~lanking the cleavage site in the substrate may form a double stranded duplex with (X) n and (X) n' as a result of base pairing, which base pairing is well known in the art tSee ~or example: Sambrook, 1989]. The formation o~ a double stranded duplex between the nucleotides may be referred to as hybridization [Sambrook, 1989]. The extent of hybridization or duplex formation between the ribozyme and its substrate can be CA 02222793 l997-l2-Ol W O ~6/109~6 PCT/AU96/00343 readily assessed, ~or example, by labeling one or both components, such as with a radiolabel, and then subjecting the reaction mixture to polyacrylamide gel electrophoresis under non-denaturing conditions [Sambrook, 1989]. If the target is cleaved speci~ically on incubation with the compound, the compound is active and ~alls within the scope o~ this invention. Accordingly, a ribozyme containing substituted or modi~ied nucleotides in the conserved region may be simply tested ~or ~n~nllclease activity in a routine m~nne~.

As will be readily appreciated by workers in the ~ield to which this invention relates, the cleavage o~ a target RNA
may be readily assessed by various methods well known in the art [See ~or example: Sambrook, 1989]. Cleavage may, ~or example, be assessed by running the reaction products (where the substrate is radioactively labeled) on acrylamide, agarose, or other gel systems under denaturing conditions, and then subjecting the gel to autoradiography or other analytical technique to detect cleavage ~ragments [Sambrook, 1989].

In another embodiment, the invention provides a composition which comprises the compounds above in association with an acceptable carrier.

The invention is also directed to an oligonucleotide trans~er vector containing a nucleotide sequence or sequences which on transcription gives rise to the compounds above. The trans~er vector may be a bacterial plasmid, a recombinant bacterial plasmid, a bacteriophage DNA, a cosmid, or an eukaryotic viral DNA. The trans~er vector may also contain an appropriate transcription promoter sequence such as that ~or RNA polymerase II, RNA polymerase III, a viral promoter such as SV40 or ~IV LTR, a plant promoter such as CaMV S35 or a promoter associated with animal health The vector may also contain an appropriate termination sequence. Preferably, the plant or animal promoter is capable of expression in a regulated manner.
Such promoter control regions would be regulated by - endogenous signals to direct either tissue specific or S temporal expression or by externally administered compounds to elicit transcription of downstream sequences. It may also contain sequences to effect integration into the host genome on episomal replication in the host cell.

The invention also provides a host cell transformed by the transfer vector as mentioned above, which may be a prokaryotic host cell or an eukaryotic host cell e.g.
yeast cell or yeast protoplast, E. coli host cell, a monkey host cell (e.g. COS), a Chinese hamster ovary host cell, a m~mm~lian host cell, a plant host cell, or a plant protoplast host cell.

In one embodiment, there is provided a packaged oligonucleotide transfer vector, as mentioned hereinabove, which is a plant virus, a composite m~mm~lian virus, a geminivirus, a Ti or Ri plasmid, an infective phage particle or portion thereo~.

In another embodiment, the composition, as discussed above, is in association with an acceptable carrier. This invention also provides a composition as discussed hereinabove wherein the oligonucleotide is an oligoribonucleotide or an RNA-DNA hybrid molecule comprising nucleotides which may be substituted or modified in their sugar, base or phosphate group. It is pre~erred that the oligonucleotide be an oligoribonucleotide or a hybrid RNA-DNA molecule. However, other substitutions or modifications in the nucleotide are possible providing that endonuclease activity is not lost. Such derivatives or modifications are described below W096/40906 CA 02222793 l997-l2-ol PCT/AU96/00343 The nucleotides may be in the ~orm of deoxyribonucleotides, ribonucleotides, deoxyribonucleotide ribonucleotide hybrids, or derivatives thereo~ as herein described. The ~lanking sequences (X) n and (X) n' may be chosen to optimize stability o~ the ribozyme ~rom degradation. For example, deoxyribonucleotides are resistant to the action of ribonucleases. Modi~ied bases, sugars or phosphate linkages o~ nucleotides, such as phosphoramidate, or phosphorothioate linkages in the sugar phosphate chain o~ Xn and Xn,, may also provide resistance to nuclease attack. Binding a~inity may also be optimized in particular circumstances, by providing nucleotides solely in the ~orm o~ ribonucleotides, deoxyribonucleotides, or combinations thereof. In some circumstances it may be necessary to optimize the composition o~ the sequences (X) n and (X) n' ~ to m~;m;ze target RNA cleavage. The cleavage activity o~ ribozymes having ~lanking nucleotide sequences which hybridize to target sequences and which are comprised wholly of deoxyribonucleotides may, in some circumstances, have reduced activity. In such circumstances optimization may involve providing a mixture o~ deoxyribonucleotides and ribonucleotides in the nucleotide sequences (X) n and (X) n' ~
For example, nucleotides in the ribozyme which are proximal to the cleavage site in a target RNA may be in the form of ribonucleotides.

The respective 3' and 5' termini o~ the sequences (X) n and (X) n' or alternatively the 3' and 5' end termini o~ the ribozyme, may be modi~ied to stabilize the ribozyme ~rom degradation. For example, blocking groups may be added to prevent terminal nuclease attack, in particular 3'-5' progressive exonuclease activity. By way of example, blocking groups may be selected from substituted or unsubstituted alkyl, substituted or unsubstituted phenyl, substituted or unsubstituted alkanoyl. Substituents may be selected ~rom Cl - C5 alkyl; halogens such as F, Cl or Br;
hydroxy; amino; C1 - C5 alkoxy and the like. Alternatively, CA 02222793 l997-l2-Ol nucleotide analogues such as phosphorothioates, methylphosphonates or phosphoramidates or nucleoside derivatives (such as alpha - anomer of the ribose moiety) which are resistant to nuclease attack may be employed as terminal blocking groups. The blocking group may be an inverted linkage such as a 3' 3' thymidine linkage or a 5' 5' pyrophosphate linkage as in the guanosine cap.

Alternatively, groups which alter the susceptibility of the rIbozyme molecule to other nucleases may be inserted into the 3' and/or 5' end of the ribozyme. For example, 9-amino-acridine attached to the ribozyme may act as a terminal blocking group to generate resistance to nuclease attack on the ribozyme molecules and/or as an intercalating agent to aid endonucleolytic activity. It will be readily appreciated that a variety o~ other chemical groups, e.g.
spermine or spermidine could be used in a related manner.

It is also possible to stabilize the ribozyme ~rom degradation by embedding it in an RNA molecule. These molecules can be produced either in vitro or in vivo by DNA
coding sequences being operably linked to transcriptional control sequences as appropriate. Examples o~ RNA molecules into which ribozymes could be inserted may include, but are not limited to, tRNA, mRNA, rRNA, snRNA or other RNA
molecules. In addition, the ribozyme may be inserted into an engineered stable stem loop structure. The compound may also be coupled with other stabilizing structures such as a transcription terminator on the 3' end such as the T7 terminator, p-independent terminator, cry element (Gel~and et al. U.S. Patent No. 4,666,848, issued May 19, 1987) or the TrpE terminator. Furthermore, sequences such as the poly(A) addition signal AATA~A may be added. In addition, strategies involving changing the length o~ the 3~ non-coding region may be used (see Gillies, U.S. Patent No.5,149,635, issued September 22, 1992). Alternatively, a stabilizing sequence or protein binding domain such as W096/40906 CA 02222793 l997-l2-ol PCT/AU96/00343 Sioud, PCT International application WO 94/10301 may be used. Further, it is possible to insert the compound into a DNA molecule as well.

The compounds o~ this invention may be covalently or non-covalently associated with a~inity agents such as proteins, steroids, hormones, lipids, nucleic acld sequences, intercalating molecules (such as acridine derivatives, ~or example 9-amino acridine) or the like to modi~y b; n~; n~ a~inity ~or a substrate nucleotide sequence or increase a~inity ~or target cells, or localization in cellular compartments or the like. For example, the ribozymes of the present invention may be associated with RNA binding peptides or proteins which may assist in bringing the ribozyme into juxtaposition with a target nucleic acid such that hybridization and cleavage o~ the target sequence may take place. Nucleotide sequences may be added to the respective 3' and 5' termini o~ the sequences (X) n and (X) n' or alternatively the 3' and 5' end termini o~
the ribozyme to increase a~inity ~or substrates. Such additional nucleotide sequences may ~orm triple helices with target sequences [Strobel, 1991] which may enable interaction with an intramolecularly ~olded substrate.
Alternatively, modi~ied bases (non-natural or modified bases as described in Principles o~ Nucleic Acid Structure [Saenger, 1984]) within the additional nucleotide sequences may be used that will associate with either single stranded or duplex DNA generating base pair, triplet, or quadruplet, interactions with nucleotides in the substrate. Suitable bases would include inosine, 5-methylcytosine, 5-bromouracil and other such bases as are well known in the art, as described, ~or example, in Principles o~ Nucleic Acid Structure [Saenger, 1984].

The compounds o~ this invention may be produced by nucleotide synthetic techniques which are known in the art, and described ~or example by Carruthers et al., Foehler et W096/40906 CA 02222793 l997-l2-ol PCT/AU96/00343 al. and Sproat et al. [Carruthers, 1987; Foehler, 1986;
Sproat, 1984]. Generally, such synthetic procedures involve the sequential coupling of activated and protected - nucleotide bases to give a protected nucleotide chain, whereafter protecting groups may be removed by suitable ~ treatment. Preferably the compounds will be synthesized on an automated synthesizer such as those made by Applied Biosystems (a Division of Perkin Elmer~, Pharmacia or Millipore. Alternatively, the ribozymes in accordance with this invention may be produced by transcription of nucleotide sequences encoding said ribozymes in host-cells or in cell free systems utilizing enzymes such as T3, SP6 or T7 RNA-polymerase.

In addition to being synthesized chemically, ribozymes with modified nucleotides may be synthesized enzymatically. The phosphodiester bonds of RNA can be replaced by phosphorothioate linkages by in vitro transcription using nucleoside ~-phosphorthiotriphosphates. T7 RNA polymerase specifically incorporates the Sp isomer of ~-phosphorthiotriphosphate with inversion of configuration to produce the Rp isomer o~ the phosphorothioate linkage. The methods to produce transcripts fully substituted with phosphorothioate linkages adjacent to a given nucleotide, or to produce partially substituted transcripts containing approximately one phosphorothioate linkage. The methods to produce transcripts fully substituted with phosphorothioate linkages adjacent to a given nucleotide, or to produce partially substituted transcripts containing approximately one phosphorothioate linkage per molecule, are described by Ruffner and Uhlenbeck (1990). Conrad et al. (1995) describe methods of using T7 RNA polymerase to produce chimeric transcripts containing ribonucleotides and deoxyribonucleotides (with and without phosphorothioate linkages), and also ribonucleotides and 2~-O-methylnucleotides (with and without phosphorothioate linkages). These methods have been shown to produce CA 02222793 l997-l2-Ol transcripts containing up to 50~ deoxyribonucleotides, and up to 58~ 2'-0-methylnucleotides. Aurup et al (1992) describe methods for using T7 polymerase to produce transcripts containing 2'-~luoro-2'-deoxyuridine, 2'-~luoro-2'-deoxycytidine, and 2'-amino-2'deoxyuridine. (Aurup, 1992; Conrad, 1995; Ru~ner, 1990) Further means ~or producing the ribozymes o~ this invention are ~urther discussed below [Sambrook, 1989].

Nucleotides represented in the compounds above comprise a sugar, base, and a monophosphate group or a phosphodiester linkage. Accordingly, nucleotide derivatives or modi~ications may be made at the level o~ the sugar, base, monophosphate groupings or phosphodiester linkages. It is pre~erred that the nucleotides in the compounds above be ribonucleotides or RNA/DNA hybrids, however, other substitutions or modi~ications in the nucleotide are possible providing that ~n~o~l~clease activity is not lost.

In one aspect o~ this invention, the sugar of the nucleotide may be a ribose or a deoxyribose such that the nucleotide is either a ribonucleotide or a deoxyribonucleotide, respectively. Furthermore, the sugar moiety of the nucleotide may be modi~ied according to well known methods in the art tSee ~or example: Saenger, 1984; Sober, 1970].
This invention embraces various modi~ications to the sugar moiety o~ nucleotides as long as such modi~ications do not abolish cleavage activity o~ the ribozyme. Examples o~
modi~ied sugars include replacement of secondary hydroxyl groups with halogen, amino or azido groups; 2~-alkylation;
con~ormational variants such as the 02'-hydroxyl being cis-oriented to the glycosyl C1' -N link to provide arabinonucleosides, and con~ormational isomers at carbon C1' to give alpha -nucleosides, and the like. In addition, the invention is directed to compounds with a substituted 2 hydroxyl such as 2' O-allyl, or 2' O-methyl. Alternatively, W096/40906 CA 02222793 l997-l2-ol PCT/AU96/00343 the carbon backbone of the sugar may be substituted such as in 2' C-allyl.

Accordingly, the base of the nucleotide may be a~n;ne, 2-amino adenine, cytosine, guanine, hypoxanthine, inosine, methyl cytosine, thymine, xanthine, uracil, or other methylated bases.

Nucleotide bases, deoxynucleotide bases, and ribonucleotide bases are well known in the art and are described, for example in Principles of Nucleic Acid Structure [Saenger, 1984]. Furthermore, nucleotide, ribonucleotide, and deoxyribonucleotide derivatives, substitutions and/or modifications are well known in the art [See for example:
Saenger, 1984; Sober, 1970], and these may be incorporated in the ribozyme made with the proviso that endonuclease activity of the ribozyme is not lost. As mentioned previously, endoribonuclease activity may be readily and routinely assessed.
In addition, a large number of modified bases are ~ound in nature, and a wide range o~ modified bases have been synthetically produced [See for example: Saenger, 1984;
Sober, 1970~. For example, amino groups and ring nitrogens may be alkylated, such as alkylation of ring nitrogen atoms or carbon atoms such as N1 and N 7 of guanine and C 5 of cytosine; substitution of keto by thioketo groups;
saturation of carbon-carbon double bonds, and introduction of a C-glycosyl link in pseudouridine. Examples of thioketo derivatives are 6-mercaptopurine and 6-mercaptoguanine.
Bases may be substituted with various groups, such as halogen, hydroxy, amine, alkyl, azido, nitro, phenyl and the like. The phosphate moiety of nucleotides or the phosphodiester linkages of oligonucleotides are also subject to derivatization or modifications, which are well known in the art. For example, replacement of oxygen with nitrogen, sulphur or carbon gives phosphoramidates, W O ~6/10306 CA 02222793 l997-l2-0l PCT/AU96/00343 (phosphorothioates, phosphorodithioates) and phosphonates, respectively. Substitutions of oxygen with nitrogen, sulphur or carbon derivatives may be made in bridging or non bridging positions. It has been well established from work involving antisense oligonucleotides [Uhlman, 1990] that phosphodiester and phosphorothioate derivatives may efficiently enter cells (particularly when of short length), possibly due to association with a cellular receptor.
Methylphosphonates are probably readily taken up by cells by virtue of their electrical neutrality.

A further aspect of the invention provides alternative linkages such as an amide, a sulfonamide, a hydroxylamine, a formacetal, a 3~-thioformacetal, a sulfide, or an ethylene glycol function to replace the conventional phosphodiester linkage. These modifications may increase resistance towards cellular nucleases and/or improved pharmacokinetics.

Information on Synthesis of Protected Nucleotides and their Incorporation into Modified Ribozymes.

POSSIBLE NnUCLEOTIDE
Modifications Suaar Modifications may be 2' fluoro, 2~ amino, 2~ O-allyl, 2' C-allyl, 2' O-methyl, 2' O-alkyl, 4'-thio-ribose, ~-anomer, arabinose, other sugars, or non-circular analogues.

Phosphate Modifications may be phosphorothioate (non-bridging), phosphorodithioate (non bridging), 3' bridging phosphorothioate, 5I briding phosphorothioate, phosphoramidates, 3' bridging phosphoramidate, 5' bridging phosphoramidate, methyl phosphonate, other alky phosphonates or phosphate triesters.

The phosphodiester linkage may be replaced by an amide, carbamate, thiocarbamate, urea, amine, hydroxylamine, formacetal, thioformacetal, allyl ether, allyl, ether, thioether, or PNA (peptide nucleic acid) linkage.

- Modifications in base may be purine, 2,6-diaminopurine, 2-aminopurine, O6-methylguanosine, 5-alkenylpyrimidines, 5-propyne, inosine, 5-methylcytosine, pseudouridine, abasic (ribose or deoxyribose).

Some nucleotides may be replaced with the ~ollowing chemical linkers: 1,3-propane-diol, alkane-diols, or various polymers of (ethyleneglycol, tetraethylene glycol, hexaethyleneglycol).
Other Modifications to the 3' end may be selected ~rom: 3'-3' inverted linkage (inverted diester or inverted phosphoramidate). 3'-3~ linked abasic ribose, or end-capped (methoxyethylamine phosphoramidate).
Modified sugars may be synthesized as follows:
2~-deoxy-2'-~luoro uridine (Sinha, 1984); 2'-deoxy-2' ~luoro cytidine (Sinha, 1984); 2'-deoxy-2' fluoroadenosine;
synthesis and incorporation into ribozyme (Olsen, 1991);
2'-deoxy-2'-amino uridine and 2'-deoxy-2'-amino cytidine (Heidenreich, 1994); 2'-O-allyl-(uridine or cytidine or adenosine or guanosine) (Available from Boehringer M~nnh~im, Mannheim, Germany) or (Badger, 1994).
2'-deoxy-2'-C-allyl-ribonucleotides; 2'-O-methyl ribonucleotides see Review: (Sproat, B.S., l991A) (also Available ~rom Chemgenes, Waltham, MA. or Glen Research, Sterling, VA. other 2'-O-alkyl-ribonucleotides, Synthesis see (Monia, B. P., 1993; Sproat, B. S , l991B); a-anomer of - uridine, cytidine, adenosine and guanosine, see (Debart, F., 1992 and references therein)i Other modified sugars, etc.

W096/40906 CA 02222793 l997-l2-0l PCT/AU96/00343 Arabinose (Garbesi, A., 1993); Hexose-thymidine (Augustyns, K., 1992) and linear analogues o~ sugars (Hendry, 1994).

Modi~ied phosphates may be synthesized as ~ollows: -Phosphorothioate; synthesized by modi~ication o~ oxidation procedure during phosphoramidite synthesis. Reagents commercially available from Perkin Elmer and others, products are mixture o~ isomers, some methods available ~or stereospeci~ic synthesis o~ phosphorothioate, see re~:
(Stec, l991); Phosphorodithioate; (Eldrup, A. B., 1994;
Caruthers, 1991; Beaton, 1991); 3'-bridging phosphorothioate; 5' bridging phosphorothioate;
phosphoramidates (non-bridging, oxidize the phosphite triester with solution containing the required amine);
(Froehler, B., 1988; Jager, A., 1988; Letsinger, R.L., 1988); 3' bridging phosphoramidate (NH replaces 3' O) (Forms very stable duplexes) (Letsinger, 1992; Gryaznov, S. M., 1995; Chen, J. K., 1995); 5' Bridging Phosphoramidate (NH
replaces 5' O; thymidine analogue only, weak binder) (Gryaznov, S. M., 1992); Methylphosphonate (reagents are commercially available; Glen Research or Chemgenes Stereospeci~ic; Rp isomers bind stronger: (Savchenko, 1994;
Miller, 1991); 5'-deoxy, 5'-methylphosphonate (Szabo, 1995);
Other alkyl-phosphonates (Fathi, 1994A; Fathi, 1994B);
Phosphate triesters(Summers, 1986).

Replacements ~or the Phosphodiester Linkage may be synthesized as ~ollows:
For review see (De Mesmaker, 1995) Amides (Chur,1993;
Blommers, 1994; De Mesmaeker, 1993; De Mesmaeker, 1994A; De Mesmaeker, 1994B; Lebreton, 1993; Lebreton, 1994A; Lebreton, 1994B; Idsiak, 1993): Carbamate (Waldner, 1994; Stirchak, 1987; Habus, 1994; Thiocarbamate (Waldner,1995); Ureas (Waldner, 1994) Amines (De Mesmaeker,1994C; Caulfield, 1994); Hydroxylamine (Debart,1992; Vasseur,1992; Formacetal (Matteucci,1990; Jones, 1993)Thio~ormacetal (Jones,1993);
Allyl ether (Cao,1994); Allyl, Ether, Thioether (Cao, W096/40906 CA 02222793 l997-l2-o1 PCT/AU96/00343 1994); Alkane (De Mesmaeker,1994; PNA A selection of binding and antisense properties (Nielsen, 1993A; Hanvey, 1992; Egholm, 1993; Nielsen, 1993B)i PNA Synthesis (Egholm, 1992A; Egholm, 1992B);Prepn of purine PNA monomers and S oligos (available commercially ~rom Millipore corporation).

Modified bases may be synthesized as follows:
Purine; synthesis and incorporation into ribozyme (Slim, 1992; Fu,1992; Fu, 1993); 7-deazaGuanosine, synthesis and incorporation into ribozyme (Fu, 1993); Inosine, synthesis and incorporation into ribozyme (Slim,1992; Fu, 1993)7-deazaAdenosine, synthesis and incorporation into ribozyme (Fu, 1992; Seela, 1993). 06-methylguanosine, synthesis and incorporation into ribozyme (Grasby, 1993);
2~6-~;~m;nopurine~ synthesis (Sproat, 1991); 2-aminopurine, synthesis and incorporation into ribozyme (Ng, 1994; Tuschl, 1993); Isoguanosine, synthesis and incorporation into ribozyme (Ng, 1994; Tuschl, 1993); Xanthosine, synthesis and incorporation into ribozyme (Tuschl, 1993);6-azathymidine, 6-aza-2'-deoxycytidine, synthesis and incorporation into oligonucleotides (Sanghvi, 1993); 5-alkenylpyrimidines;
5-propyne (Gilead, Froehler); inosine; 5-methylcytosine;
pseudouridine; abasic ribose or deoxyribose.

STJMMAR~ OF NUCL~O~ MODIFICATIONS WHICH HAVE BEEN TESTED
IN RIBOZYMES.
Sugars.
Modi~ications may be made to the 2'OH group o~ the sugar at all non-conserved nucleotides; modi~ications tested have been 2'H (DNA), 2'F, 2'amino, 2'-O-allyl, 2'-O-methyl, 2'-C-allyl.
Selected modi~ications may be made to the 2'OH groups of the conserved nucleotides C3, U4, A6, N7, A9, G12, A13, A14, N15.2.
Modi~ications cannot be made to the 2'0~ groups o~ G5, G8 and A15.1.

CA 02222793 1997-12-01 ~ A~
W O 96/40906 PCT/AU .

For a ribozyme with good cleavage activity, modifications should not be made to G5, A6, G8, G12, A15.1 (except G12 can be 2'H (DNA)).
Generally, except ~or modifications at G5, G8 and A15.1, no 5 single modification causes a big reduction in cleavage activity; however, activity decreases as more modifications are included in the ribozyme.

Phosphates.
10 The phosphate groups of the nonconserved nucleotides may be phosphorothioates (phosphorothioated DNA). Pre~erably, when non-conserved nucleotides are DNA, only two or three phosphates at the 3' and 5' ends oi~ the ribozyme are phosphorothioates.
15 The phosphates 5' to the conserved nucleotides C3, U4, G5, G8 and G12, and 3' to A9 and N15.2, may be phosphorothioates; but phosphates 5' to A9, A13 and A14 may not be phosphorothioates.

Conserved nucleotides.

Sugar - 2'-OH group can be modi~ied (except, probably, ~or 2'amino). 2'H (Yang, 92), 2'F (Pieken, 91; Heidenreich, 92), 2'-0-allyl (Paolella, 92), 2~-0-Methyl (Usman, 95) are all modifications that permit cleavage.
Possibly cannot have 2'amino modification (several Cs in ribozyme had 2'amino modii~ication which resulted in reduction in activity, and effect is probably due to 2'amino on C3 and/or C15.2) (Pieken, 91).
Phosphate - 5~ phosphate can be phosphorothioate (Shimayama, 93) Sugar - 2~0H group can be modified. 2'H (Yang, 92), 2'F
(Pieken, 91; Heidenreich, 92), 2'amino (Pieken, 91), 2'-C-allyl (Usman, 95), 2'-0-allyl (but keep as 2'0H i~ A6 is 2'-0-allyl) (Paolella, 92) are modifications that permit cleavage.

W O ~6/lC9~ PCT/AU96/00343 Phosphate - 5' phosphate can be phosphorothioate (Shimayama, 93) ~ Base - 2-amino group on G base is essential (cannot be inosine) (Oddi, 90; Fu, 92) Sugar - cannot make modifications to 2'OH of G5. Cannot have 2'H (Perreault, 90; Perreault, 91; Fu, 92; Williams, 92), 2'amino (Pieken, 91; Williams, 92), 2'-0-methyl (Paolella, 92), 2'F (Williams, 92).

Base - can be purine (i.e. 6-amino group is not essential) (Fu, 92). N7 cannot be C7 in A base (Fu, 92).
Sugar- 2'0H group can be modified. 2'H (Perreault, 90i Olsen, 91; Yang, 92; Fu, 92), 2'F (Olsen, 91), 2~-0-allyl (but only if U4 is 2'0H) (Paolella, 92) are modifications that permit cleavage.

Seems to be a sensitive site for pyrimidine endonucleases;
protection achieved if rN is rG or rA (Shimayama, 93).
Sugar - 2~0H group can be modified. 2'H (tested dT) (Yang, 92), 2~F (Pieken, 91; Heidenreich, 92), 2'-amino (Pieken, 91), 2'-0-allyl (Paolella, 92), 2'-0-Methyl (Usman, 95) are all modifications that permit cleavage.
3' phosphate can be phosphorothioate (has been tested for N
= U) (Shimayama, 93).

Sugar - cannot make modifications to 2'OH of G8. Cannot have 2'H (Fu, 92; Williams, 92; Yang, 92), 2'F (Williams, 92), 2~amino (Williams, 92), 2'-0-allyl (Paolella, 92).
(Perreault (91) says can have 2'H, but Yang (92) says this site is critical if lots of other conserved nucleotides are DNA.) Phosphate - 5~ phosphate probably can be phosphorothioate (see N7 phosphate).

Sugar - 2~0H group can be modi~ied. 2'H (Olsen, 91; Fu, 92;
but Perreault (91) says cannot be 2'H), 2~F (Olsen, 91;

Wos~409C6 CA 02222793 l997-l2-ol PCT/AU96/00343 Pieken, 91), 2'-O-allyl (Paolella, 92), 2'-O-Methyl (Usman, 95) are all modifications that permit cleavage.
Phosphate - 5' phosphate cannot be phosphorothioate (Buzayan, 90; Ru~fner, 90). 3' phosphate can be phosphorothioate (Shimayama, 93).

Base - 2-amino group is essential (cannot be inosine) (Sli~, 92).
Sugar - 2'OH group can tolerate some modifications. 2~H
(Perreault, 91; Yang, 92; Williams, 92), 2'amino (Pieken, 91; Williams, 92) are OK. Cannot be 2'F (Williams, 92), 2'-O-allyl (Paolella, 92).
Phosphate - 5' phosphate can be phosphorothioate (Shimayama, 93).

Base - Can change N7 to C. in A base (Fu, 92). 6-amino group essential (cannot be purine) (Slim, 92).
Sugar - 2'OH group can tolerate some modifications. 2'H
(Perreault, 91; Yang, 92), 2'-O-allyl (Paolella, 92), 2'-O-Methyl (Usman, 95) are modifications that permit cleavage. Cannot have 2'F i~ each of A13, A14, A15.1 have 2'F (Pieken, 91).
Phosphate - 5' phosphate cannot be phosphorothioate (Ru~ner, 90).

Base - Can change N7 to C. (Fu, 92). Can be purine (Slim, 92).
Sugar - 2'OH group can tolerate some modi~ications. 2'H
(Perreault, 91; Yang, 92), 2'-O-allyl (Paolella, 92), 2'-O-Methyl (Usman, 95) are modi~ications that permit cleavage. Cannot have 2'F i~ each o~ A13, A14, A15.1 have 2'F (Pieken, 91).
Phosphate - 5' phosphate cannot be phosphorothioate (Ruffner, 90).
A15.1 Base - Can change N7 to C. (Fu, 92). 6-amino group essential (cannot be purine) ~Slim, 92).

CA 02222793 1997-12-01 PCT/AU9~'00 Sugar - Cannot modify 2'0H. Cannot have 2'H (Yang, 92), 2'-0-allyl (Paolella, 92), 2'F (i~ A13 and A14 also are 2'F) (Pieken, 91).
N15.2 5 Sugar - selected modifications permit cleavage. 2'F
(Pieken, 91; Heidenreich, 92), 2'-0-allyl (Paolella, 92), 2'-0-Methyl (Usman, 95) are modifications that permit cleavage.
Rates are low if 2'H (Yang, 92). Possibly cannot have 10 2'amino modification (several Cs in ribozyme had 2'amino modification which resulted in reduction in activity, and ef~ect is probably due to C3 and/or C15.2) (Pieken, 91).
3' phosphate can be phosphorothioate (Shimayama, 93) Modification~ at the 3' end of an oligonucleotide or ribozyme.
3'MEA(methoxyethylamine)phosphoramidate in last two (or last) internucleotide linkages; 3'-3' inverted diester linkage or 3'-3' inverted phosphoramidate (Shaw, 91).
3'-3' inverted Thymidine, or 3'-3' linked abasic ribose (Usman, 95).

Any combination o~ the above listed nucleotide modifications, substitutions, or derivatizations, made at the level o:~ the sugar, base, or monophosphate groupings or phosphophodiester linkages may be made in the compounds provided that endonuclease activity is not lost.

The compounds of this invention may be incorporated and expressed in cells as a part of a DNA or RNA trans~er ~ector, or a combination thereoi~, for the maintenance, replication and transcription of the ribozyme sequences of this invention.

Nucleotide sequences encoding the compounds o~ this invention may be integrated into the genome of a eukaryotic or prokaryotic host cell f~or subsequent expression (~or example as described [Sambrook, 1989~). Genomic integration may be facilitated by transfer vectors which integrate into the host genome. Such vectors may include nucleotide sequences, for example of viral or regulatory origin, which facilitate genomic integration Methods for the insertion of nucleotide sequences into a host genome are described ~or example in Sambrook et al. and Hogan et al. [Sambrook, 1989;
Hogan, 1986; 1989].

Nucleic acid sequences encoding the ribozymes of this invention integrated into the genome may preferably include promoter and enhancer elements operably linked to the nucleotide sequence encoding the ribozyme of this invention, with an appropriate termination sequence and capable of expressing said ribozyme in a eukaryotic (such as ~n ~ m~ 1 or plant cells) or prokaryotic (such as bacteria) host cells.
Ideally, the promoter and enhancer elements are designed for expression in a tissue and/or developmentally specific m~nner, Additionally, the compounds of the present invention may be prepared by methods known per se in the art for the synthesis of RNA molecules. (For example, according to recommended protocols of Promega, Madison, Wis., USA). In particular, the ribozymes of the invention may be prepared from a corresponding DNA sequence (DNA which on transcription yields a ribozyme, and which may be synthesized according to methods known per se in the art for the synthesis of DNA) operably linked to an RNA polymerase promoter such as a promoter for T3 or T7 polymerase or SP6 RNA polymerase. A DNA sequence corresponding to a ribozyme of the present invention may be ligated into a DNA transfer vector, such as plasmid or bacteriophage DNA. Where the transfer vector contains an RNA polymerase promoter operably linked to DNA corresponding to a ribozyme, the ribozyme may be conveniently produced upon incubation with an RNA
polymerase. Ribozymes may, therefore, be produced in vitro W 096/40906 CA 02222793 l997-l2-Ol PCT/AU~G,'Cr3A~

by incubation of RNA polymerase with an RNA polymerase promoter operably linked to DNA encoding a ribozyme, in the presence o~ ribonucleotides and an appropriate buf~er. In vlvo, prokaryotic or eukaryotic cells (including m~mm~l ian, plant and yeast cells) may be trans~ected with an appropriate trans~er vector containing genetic material encoding a ribozyme in accordance with the present invention, operably linked to an RNA polymerase promoter such that the ribozyme is transcribed in the host cell.
Transfer vectors may be bacterial plasmids or viral (RNA and DNA) or portion thereo~. Nucleotide sequences corresponding to ribozymes are generally placed under the control o~
strong promoters such as, the lac, SV40 late, SV40 early, metallothionein, or lambda promoters. Particularly use~ul are promoters regulated in a tissue or a temporal (developmental) speci~ic manner or tightly regulated inducible promoter suitable ~or gene theory, which may be under the control of exogenous chemicals. The vector may be an adenovirus or an adeno-associated virus. See ~or example PCT International Publication No. WO 93/03769, "Adenovirus Mediated Trans~er o~ Genes to the Gastrointestinal Tract", U.S. Serial No. 747,371; PCT International Publication No.
WO 94/11506, "Adenovirus-Mediated Gene Trans~er to Cardiac and Vascular Smooth Muscle," J. Leiden et al., U.S. Serial No. 07/977,496; PCT International Publication No. W0 94/11522, IlVector :Eor the Expression oi~ Therapy-Relevant Genes," U. Stein et al.,PCT International Publication No. WO
94/11524, "Targetable Vector Particles," W. Anderson et al., U.S. Serial No. 973,307;
PCT International Publication No. W0 94/17832, "Targeting and Delivery o~ Genes and Antiviral Agents into Cells by the Adenovirus Penton," G. Nemerow et al., U.S. Serial Nos.
08/046,159 and 08/015,225. Ribozymes may be directly transcribed in vivo ~rom a trans~er vector, or alternatively, may be transcribed as part o~ a larger RNA
molecule. For example, DNA corresponding to ribozyme sequences may be ligated into the 3' end o~ a reporter gene, W O ~G11C9~ CA 02222793 l997-l2-Ol PCT/AU~G~

for example, a~ter a translation stop signal. Larger RNA
molecules may help to stabilize the ribozyme molecules against nuclease digestion within cells. On translation the reporter gene may give rise to a protein, possibly an enzyme whose presence can be directly assayed.

The compounds o~ this invention may be involved in gene therapy techniques, where, ~or example, cells ~rom a human su~fering ~rom a disease, such as HIV, are removed ~rom a patient, treated with the ribozyme or transfer vector encoding the ribozyme to inactivate the infectious agent, and then returned to the patient to repopulate a target site with resistant cells, so called ex v1vo therapy. In the case o~ HIV, nucIeotide sequences encoding ribozymes of this invention capable of inactivating the HIV virus may be integrated into the genome o~ lymphocytes or may be expressed by a non-integrating vector such as adenovirus.
Such cells would be resistant to HIV in~ection and the progeny thereo~ would also con~er such resistance.
A transfer vector such as a bacterial plasmid or viral RNA
or DNA or portion thereo~, encoding one or more o~ the compounds, may be trans~ected into cells o~ an organism in vivo [See for example: Llewellyn, 1987; ~n~h~n, 1983]. Once inside the cell, the transfer vector in some cases may replicate and be transcribed by cellular polymerases to produce ribozyme RNAs which may have ribozyme sequences oi~
this invention; the ribozyme RNAs produced may then inactivate a desired target RNA. Alternatively, a trans~er vector containing one or more ribozyme sequences may be trans~ected into cells by electroporation, P~G, high velocity particle bombardment or lipofectants, or introduced into cells by way of micromanipulation techniques such as microinjection, such that the trans~er vector or a part thereo~ b~com~ integrated into the genome of the host cell.
Transcription o~ the integrated genetic material gives rise to ribozymes, which act to inactlvate a desired target RNA.

CA 02222793 l997-l2-Ol W O 96t40906 PCT/AU96/00343 Transfer vectors expressing ribozymes of this invention may be capable of replication in a host cell for stable expression of ribozyme sequences. Alternatively, transfer vectors encoding ribozyme sequences of this invention may be incapable of replication in host cells, and thus may result ~ in transient expression o~ ribozyme sequences. Methods for the production of DNA and RNA transfer vectors, such as plasmids and viral constructs are well known in the art and are described for example by Sambrook et al. [Sambrook, 1989].

Transfer vectors would generally comprise the nucleotide sequence encoding the ribozyme of this invention, operably linked to a promoter and other regulatory sequences required :Eor expression and optionally replication in prokaryotic and/or eukaryotic cells. Suitable promoters and regulatory sequences for transfer vector maintenance and expression in plant, animal, bacterial, and other cell types are well known in the art and are described ~or example in Hogan [Hogan, 1986; 1989].

The ribozymes o~ the present invention have extensive therapeutic and biological applications. For example, disease causing viruses in man and animals may be inactivated by administering to a subject in~ected with a virus, a ribozyme in accordance with the present invention adapted to hybridize to and cleave speci~ic RNA transcripts of the virus. Such ribozymes may be delivered by parenteral or other means o~ administration. Alternatively, a subject infected with a disease causing virus may be administered a non-virulent virus such as vaccinia or adenovirus which has been genetically engineered to contain DNA corresponding to a ribozyme operably linked to an RNA promoter, such that the ribozyme is transcribed in the cells of the host animal, trans~ected with the engineered virus, to effect cleavage and/or inactivation o~ the target RNA transcript o~ the disease causing virus.

The ribozymes o~ the present invention have particular application to viral diseases caused ~or example, by the herpes simplex virus (HSV) or the AIDS virus (HIV). Further examples o~ human and ~n; m~ 1 disease which may be treated with the ribozymes o~ this invention include psoriasis, cervical preneoplasia, papilloma disease, bacterial and prokaryotic in~ection, viral in~ection and neoplastic conditions associated with the production o~ aberrant RNAs such as occurs in chronic myeloid leukemia. Diseases or in~ections which may be treated in plants with ribozymes of this invention include ~ungal infection, bacterial in~ections (such as Crown-Gall disease) and disease associated with plant viral in~ection. O~ particular interest would be compounds targeting genes associated with male gametophyte development. Examples include PCT
International Publication No. WO 92/18625, entitled "Male-Sterile Plants, Method For Obt~;n;ng Male-Sterile Plants And Recombinant DNA For Use Therein"; U.S. Patent No. 5,254,802, entitled "Male Sterile Brassica Plants," S. Hoekstra et al.;
PCT International Publication No. WO 93/25695, entitled "Maintenance o~ Male-Sterile Plants," M. Williams et al., claiming the priority o~ U.S. Serial Nos. 07/970,840 and 07/899,072; PCT International Publication No. WO 94/25593, entitled "Method For Obt~;n;ng Male-Sterile Plants" Stiekema et al.; PCT International Publication No. WO 94/29465, entitled "Process For Generating Male Sterile Plants" Dirks et al.

The period o~ treatment would depend on the particular disease being treated and could be readily determined by a physician or by a plant biologist as appropriate. Generally treatment would continue until the disease being treated was ameliorated.

The ribozymes o~ the present invention also have particular application to the inactivation o~ RNA transcripts in bacteria and other prokaryotic cells, plants, ~n; m~ s and yeast cells. In bacteria, RNA transcripts of, for example, bacteriophage, (which cause bacterial cell death) may be inactivated by trans~ecting a cell with a DNA transfer vector which is capable of producing a ribozyme in S accordance with the present invention which inactivates the ~ phage RNA. Alternatively, the ribozyme itsel~ may be added to and taken up by the bacterial cell to ef~ect cleavage o-the phage RNA. Similarly, eukaryotic and prokaryotic cells in culture may, for example, be protected from infection or disease associated with mycoplasma infection, phage infection, fungal infection and the like.

RNA transcripts in plants may be inactivated using ribozymes encoded by a transfer vector such as the Ti plasmid o~
Aarobacterium tumefaciens. When such vectors are transfected into a plant cell and integrated, the ribozymes are produced under the action of RNA polymerase and may effect cleavage of a specific target RNA sequence.
Endogenous gene transcripts in plants, ~n; m~ or other cell types may be inactivated using the compounds of the present invention. Accordingly, undesirable phenotypes or characteristics may be modulated. It may, ~or example, be possible using the ribozymes o~ the present invention to remove stones ~rom ~ruit or treat diseases in hllm~n~ which are caused by the production of a deleterious protein, or over production of a particular protein. The compounds described above may be used to effect male sterility by destroying the pollen production in a plant. Furthermore, for the ln vivo applications of the ribozymes of this invention in humans, animals, plants, and eukaryotic and prokaryotic cells, such as in phenotypic modification and the treatment of disease, it is necessary to introduce the ribozyme into cells whereafter, cleavage of target RNAs takes place. In vivo applications are highly suitable to the compounds as discussed herein.

W096/40906 CA 02222793 l997-l2-o1 PCT/AU96/00343 Methods ~or the introduction o~ RNA and DNA sequences into cells, and the expression o~ the same in prokaryotic and eukaryotic cells are well known in the art ~or example as discussed by Cotten and Friedman [Cotten, 1990; Friedman, 1989] The same widely known methods may be utilized in the present invention.

The compounds o~ this invention may be incorporated into cells by direct cellular uptake, where the ribozymes o~ this invention would cross the cell membrane or cell wall ~rom the extracellular environment. Agents may be employed to enhance cellular uptake, such as liposomes or lipophilic vehicles, cell permeability agents, such as dimethylsul~oxide, and the like.
The compounds o~ the present invention may be combined with pharmaceutically and veterinarally acceptable carriers and excipients which are well known in the art, and include carriers such as water, saline, dextrose and various sugar solutions, ~atty acids, liposomes, oils, skin penetrating agents, gel ~orming agents and the like, as described ~or example in Remington's Pharmaceutical Sciences, 17th Edition, Mack Publishing Co., Easton, Pa., Edited by Ostol et al., which is incorporated herein by re~erence.
Agriculturally acceptable carriers and excipients are well known in the art and include water; sur~actants; detergents;
particularly biodegradable detergents; talc; inorganic and/or organic nutrient solutionsi mineral earths and clays;
calcium carbonate; gypsum; calcium sul~atei ~ertilizers such as ammonium sul~ate, ~mm~nium phosphate, urea, carborundum, and aarobacterium tume~aciens; and natural products o~
vegetable origin such as, ~or example, grain, meals and ~lours, bark meals; and the like.
The compounds o~ this invention may be provided in a composition with one or more anti-viral, anti-~ungal, W096/40906 CA 02222793 l997-l2-o1 PCT/AU96/00343 anti-bacterial, anti-parasitic, anti-protazoan or antihelminthic agents, herbicides, pesticides or the like, for example as described in the Merck Index (1989) 11th Edition, Merck & Co. Inc.

- By way of example only, therapeutic compositions of this invention may be directed against Herpes Simplex virus types 1 and 2, psoriasis, cervical preneoplasia, papilloma disease, and bacterial and prokaryotic infection. Such treatments may, for example, involve topical application of ribozyme to the site of disease. For example, in the treatment of Herpes virus lesions, ribozymes may be formulated into a cream cont~i ni ng a concentration of 0.1 nM
to 100 mM ribozyme, preferably 1 nM to 1 mM. The cream may then be applied to the site of infection over a 1 to 14 day period in order to cause amelioration of symptoms of the infection. Prior to the final development of topical formulations for the treatment of virus infection, effectiveness and toxicity of the ribozymes and formulations involving them may, for example, be tested on an animal model, such as scarified mouse ear, to which virus particles, such as 2 X lo6 plaque forming units are added.
A titer of infectious virus particles in the ear after treatment can then be determined to investigate effectiveness of treatment, amount of ribozyme required and like considerations. Similar investigations in animal models prior to human trials may also be conducted, for example, in respect of the treatment of psoriasis, papilloma disease, cervical preneoplasia, and in diseases such as HIV
infection, bacterial or prokaryotic infection, viral infectlon and various neoplastic conditions, which neoplastic conditions involve a deleterious RNA species.

Compositions for topical application are generally in the form of creams, where the ribozymes of this invention may be mixed with viscous components. The compounds of this invention may be incorporated into liposomes or other W096/40906 CA 02222793 l997-l2-ol PCT/AU96/00343 barrier type preparations to shield the ribozymes ~rom nuclease attack or other degradative agents (such as nucleases and adverse environmental conditions such as W
light).

Compositions may be provided as unit dosages, such as capsules (~or example gelatin capsules), tablets, suppositories and the like. Injectable compositions may be in the ~orm of sterile solutions o~ ribozyme in saline, dextrose or other media. Compositions ~or oral a~m;n;~tration may be in the ~orm o~ suspensions, solutions, syrups, capsules, tablets and the like. Ribozymes may also be provided in the ~orm o~ an article ~or sustained release, impregnated bandages, patches and the like. The compounds o~ this invention may be embedded in liposomes or biodegradable polymers such as polylactic acid.
Pharmaceutical compositions which may be used in this invention are described, ~or example, in Remington's Pharmaceutical Sciences, see above.
The present invention is ~urther directed to a plant DNA
expression cassette comprising a gene sequence ~lanked by promoter and terminator sequences at its 5'- and 3'ends respectively wherein said genetic sequence on expression provides a ribozyme RNA. The DNA cassette may ~urther be part o~ a DNA trans~er vector suitable ~or trans~erring the DNA cassette into a plant cell and insertion into a plant genome. In a most pre~erred embodiment o~ the present invention, the DNA cassette is carried by broad host range plasmid and which is capable o~ trans~ormation into plant cells using Aqrobacterium comprising Ti DNA on the le~t and right borders, a selectable marker ~or prokaryotes, a selectable marker for eukaryotes, a bacterial origin o~
replication and optional plant promoters and terminators such as pGA~70. The present invention also includes other means o~ trans~er such as genetic bullets (e.g. DNA-coated tungsten particles, high-velocity micro projectile bombardment) and electroporation amongst others [Maliga, 1993; Bryant, 1992; or Shimamoto, 1989].

The present invention is also directed to a transgenic plant resistant to a virus, its genome containing a sequence which gives rise, on transcription, to the nucleic acid molecule mentioned above. This transgenic plant, including fruits, and seeds thereof, may be from alfalfa, apple, arabidopsis, barley, bean, canola (oilseed rape), cantaloupe, carnation, cassava, casuarina, clover, corn, cotton, courgette, cucumber, eucalyptus, grape, melon, papaya, pepper, potato, rice, rose, snap dragon, soybean, squash, strawberry, sunflower, sweet pepper, tobacco, tomato, walnut, wheat or zucchini. Also included are the plant cells transformed by the above-mentioned transfer vector, as well as a prokaryotic, eukaryotic or yeast, plant or animal cell, comprising a nucleotide sequence which is, or on transcription gives rise to, the nucleic acid molecule.

The present invention will now be illustrated by way of non-limiting Examples only, with reference to the following non-limiting Examples, and Figures.

Example 1 Minizymes containing the deoxyribonucleotides d (GTTTT) and d(~~ ) between the conserved nucleotides A~ and G12 o~er the ~ollowing advantages:

(i) These minizymes show ~ast cleavage rates in vitro. See ~ollowing data ~or the test systems Interleukin-2, TAT, CAT, and TNF~.
(ii) The CAT minizyme (CATMgtttt) shows activity against CAT in CHO cells (see Example 2).
(iii) The IL2 minizyme (IL2Mgtttt) shows activity against Interleukin-2 in PBMN cells (see Experiment 3)(Seq ID No.
5).

Minizyme g t t t t 3' ..... xx~xxCA xxxxxxx.......... 5 A/ ~CUG
t~12 ~A~GU

- t-t-t~

1. Method for det~rm;n;ng rates o~ cleavage (kd~) by minizymes.

In these experiments, conditions are optimized so that the rate-limiting step in the reaction is cleavage o~ the substrate. The substrate consists of only a small number o~
nucleotides, in order to prevent strong sel~-association, and hence substrate association with the minizyme is not rate-limiting. In addition, the minizyme concentration is at least two-~old greater than substrate concentration, which is high enough to ensure all substrate molecules are bound by minizymes. Thus, the measured rate in these experiments should be rate o~ cleavage o~ the substrate.

The substrate is labelled on its 5' end with [32p] -phosphate.
In general, the minizyme and substrate are heated together in bu~rer ~or two minutes at 80 C without magnesium, in W O ~ 9C~ CA 02222793 1997-12-01 PCT/AU96/00343 order to denature the nucleic-acid molecules; however, this step has been shown not to be necessary in a number of cases. The cleavage reaction is initiated by adding Mg'+ to the mixture at 37 C. [MgCl2] = lOmM, [Tris.HCl buffer] = 50 mM, [Minizyme] = 5 ~M (typically), [Substrate] = 2 ~M
(typically), temperature = 37 C, pH 8.2 (for Interleukin-2 and TAT systems) and pH 8.0 (for CAT and TNF~ systems).
Samples are taken from the reaction mixture at various times, and the reaction is stopped by adding excess EDTA and formamide. The samples are electrophoresed on a polyacrylamide gel containing 7M urea, and the amounts of 5'-product and uncleaved substrate are quantified using a PhosphorImager (Molecular Dynamics) and ImageQuant software.
Kinetic parameters are obtained by fitting the data for ~ of product formed (Pt) versus time (t) to the equation Pt = P~ - (eXp(-kobst)p~) where Pt is the amount of product at time t, P~ is the amount of product at t = ~, kobs is the first-order rate-constant for the reaction, and P~ is the difference between the percentage of product at t = ~ and t = 0. This is a conventional first-order kinetic equation from which kob5, P~ a~ P~are determined by least-squares fitting of the data.

2. Sequences of molecules.

Upper-case= letters represent ribonucleotides, lower-case letters represent deoxyribonucleotides.

Interleukin-2 system. (Seq ID No. 6-10) Substrate (15-mer) 5/ UCCUGUC W GCA W g 3' W096/40906 CA 02222793 l997-l2-0l PCT/AU96/00343 Minizyme g t t t t 3' a-g-g-a-CA a-a-c-g-t-a-a-c 5' A CUG ~
A A
G AGU
~ g t-t-t Minizyme dg12dcl5-2 3'a-g-g-a-c 15-Z A a-a-c-g--t-a-a-c 5' ,A ~CUG
A \A
gl2 AGU~
~t-t-t Minizyme c c t t 3' a-g-g-a-C~ a-a-c-g-t-a-a-c 5' A 'CUG \
A A
G AGC
~ t c /
Other interleukin-2 minizymes. "x" represents the deoxyribonucleotides ~orming the linker between the ribonucleotides. The various sequences of "x" that have been tested appear in the ~ollowing table (Table 1).
3' a-g-g-a-CA a-a-c-g-t-a-a-c 5' A ~CUG~
G AGU~
x TAT system. (Seq ID No. 11-13) 40Substrate (13-mer) 5' GGAAGUC AGCCUa 3' Minizyme g t t t t 3' a-g-g-t-c-c-t_t-CA t-c-g-g-a-t-c-c-t-g 5' A ~ CUG
G
AGU
t\ g t-t-t~
Minizyme t t t t 503' a-g-g-t-c-c-t-t-CA t-c-g-g-a-t-c-c-t-g 5' A 'CUG
tG t,AG~J' \ t -t /

W 0 ~6MC9v6 CA 02222793 1997-12-01 PCT/AU96/00343 CAT 3ystem. (Seq ID No.14-17) Substrate (17-mer) 5' UUCCAUGUC GGCAGAAt 3' 5 Minizyme g t t t t 3'a--a--g-g-t--a-CA c-c-g-t--c-t-t--a 5' A CUG
A ,A
- G AGU
t g ~t-t-t'' Minizyme t t t t 3' a--a-g-g-t-a-~ c--c-g-t--c-t-t-a 5' A CUG~
A - A
G ,AGU
\t - t /

Minizyme g g t t t 3' a-a-g-g-t-a-CA c-c-g-t-c-t-t-a 5' A CUG
A A
G ,AGU
t g - t-t-g~
Tumor Necrosis Factor ~(TNFa) system. (Seq ID No. 18-20) Substrate (20-mer) 5' CCAGGCAGUC AGAUCAUCUt 3' Minizyme g t t t t 3' g-g-t-c-c-g-t-CA~ t-c-t-a-g-t-a-g-a-a 5' A, ~CUG
G ~AG~
\t-t-t~
Minizyme t t t t 3' g-g-t-c-c-g-t-CA~ t-c-t-a-g-t-a-g-a-a 5' A CUG
A ,A
C ,AGU
- t -t /

W O ~C/109~6 PCT/AU96/00343 3. Observed CIE,dV ~ e rate (kob5 min~l) and exte~t of cleavage ~oP ) of short sul~ les by ~i~ylll~S with various linkers. ("Table lB)".

Experimental con~liti~nc: 10mM MgCl~, 50 mM Tris.HCl, pH 8.2 (for Interleukin-2 and TAT systems) and pH 8.0 (for CAT and TNFa: systems), 37~C, [Substrate]= 2~M, [Min~yme]= 5,uM (except [TNCMtttt, TNFMgtttt] = 4.3~M).

Expt.1 Expt.2 Expt.3 Mean Mini~me linker kobs %P_ kobs %P_ k~b5 %P_ kob5 (6) %P_ (o~
~t.r- ' 2 S'x3' CCtt 0.010 80# 0.011 80# 0.01~ (.001) 80X
tttt 0.052 81.1 0.049 84.2 0.051 (.002) 83 (2) ttttt 0.058 86.1 0.064 82.1 0.061 (.004) 84 (3) ttttC 0.016 76.3 0.015 80.0 0.016 (.001) 78 (3) gttt 0.118 82.3 0107 84.5 0.113 (.008) 83 (2) gtttt 0.299 83.5 0.287 85.3 0.225 84.9 0.270 (.040) 85 (1) gttttt 0.287 82.4 0.253 84.1 0.270 (.024) 83 (1) gtttttt 0.200 84.3 0.169 88.6 0.185 (.022) 86 (3) gttt a 0.026 85.9 0022 94.7 0.024 (.003) 90 (6) gtttt a 0.047 81.0 0038 88.4 0.043 (.006) 85 (5) gtttg 0.12~ 82.3 0.092 89.80.107 83.9 0.107 (.015) 85 t4) gttttg 0.101 82.1 0.094 84.2 0.098 (.005) 83 (2) ggttt 0.049 82.4 0.048 89.4 0.049 (.001) 86 (5) gtgtt 0.178 83.0 0.181 83.2 0.180 (.002) 83 (1) ~ttgt 0.065 93 9 0.072 85.3 0.069 (.005) 90 (6) gtttt(dg'2dc'52) 0.041 90.0 3.040 91.7 0041 (.001) 91 (1) Sl~S~ u l~ SHEET (Rule 26) CA 02222793 lss7-l2-ol Expt.l Expt.2 Expt.3 Mean M ~zvme ~nker k~5 %P, k~5 %P_ k~5 %P~ k~5 (o) %P~ (~) TAT
tttt 0.074 89.1 0.067 91.9 0.071 (.005) 91 (2) gtttt 0.179 89.7 0.190 90.8 0.185 (.008) 90 tl) C~T
tttt 0.186 82.9 0.175 84.7 0.181 (.008) 81 (1) g t t t t~ 0.526* 60.8 0.448* 63.8 0.478* 62.8 0.48* (0.04) 62 (2) ~tttt~ 0.791* 72.0 0.809* 71.4 0.80* (0.01) 72 (1) g t t t t~ 0.650* 63.0 g t t t t~ 0.453* 61.0 g t t t t~~ 0.59* (0.16) 65 (5) ggttt 0.278* 68.8 0.352* 67.6 0.32* (0.05) 68 (1) TNF~
tttt 0.002 70.0# 0.002 70.0# 0.002 (.001) 70.0#
gtttt 0.274 59.0 0.180 74.2 0.303 58.7 0.25 (0.06) 64 (9) # fixed at this value.
~ four different s~rntheses, ~~' average of data for the four a~~
reaction is biphasic; rate constant for the initial faster reaction is given.

SUBSTITUTE SHEET (RULE 26) W 0 ~6/40906 CA 02222793 1997-12-01 PCT/AU96/00343 4. Minizymes wi~ch 5'd(~11-1-1) linkers have ~..I~LUV~d cleavage activity in vitro co~ d with those wi~h S'd(l'I~I~ linkers.

Table 2 System k~5 (Mgmt) kOb5 (Mtm) kob5 (Mgtttt)/kob;(Mtttt) Interleukin-2 0.270 O.OSl 5.3 TAT 0.185 0.071 2.6 CAT O.S9 0.181 3.3 TNFa 0.25 0.002 125 The data in table 2 show that the minizymes with gtttt linkers consistently show k~ values o~ O.2 min~1 or better. Since we know that I~2Mgtttt, with kdX = ~.27 min~1, is active in cells (see Example 3), we can conclude that a minizyme with at least this level of activity in an in vitro system should not be h;n~red in cells by its k~ value (i.e. rate o~ cleavage should not be rate limiting in cells), all other things being equal (such as target site being accessible) Example 2. ~inizyme Suppression of CAT ~xpression in C~O Cell~.

Introduction Minizymes are sequence speci~ic RNA endonucleases derived ~rom standard h~mm~rhead ribozymes by elimination of helix II
Minizymes have been shown to exhibit signi~icant in vitro cleavage activity against both short RNA targets as well as long transcribed RNA. This repo_t describes the testing o~ a particular minizyme targeted against the mRNA o~ CAT
(Chloramphenicol acetyl transferase) expressed in a m~mm~lian cell line. The minizyme is a chimeric DNA/RNA molecule synthesized by solid phase methods and trans~ected into a CHO
(chinese hamster ovary) cell line stably expressing CAT.

SUBSTITUTE S~EET ~RULE 26~

W O ~ 9C6 CA 02222793 1997-12-01 PCT/AU96/00343 -~3-Experimental Protocol A CH0 based CAT expressing cell line MC 11, in which CAT is expressed from the Human metallothionein IIA (MT) promoter, was used in all experiments. The MT promoter is transcriptionally active at very low metal concentration and a reasonable level of CAT expression is observed in the absence of induction by added metals.

8 x 104 cells were plated out in EMEM containing 10~ foetal calf serum and allowed to attach overnight (14-16 hours). The cells were washed once with 1 x PBS to remove serum, then the test molecules (pre-treated for 30 minutes with l~L of lipofectamine (GIBC0 BRL, Life Technologies, Maryland, USA) in serum-free EMEM) were transferred to the cells. The final concentration of test molecules was 10 ~M. After four hours both serum and Alamar Blue (Alamar Bio-Sciences Inc, Sacramento CA) (each to a final concentration of 10~) was added to the cells and incubation continued for a further 18 hours. At this time the supernatant was ~el..oved and the cumulative cell metabolic activity determined by measuring the extent of reduction of the Alamar Blue reagent.
The cells were harvested and CAT activity assayed (Sleigh, 1986).

S~J8SmUTE SH~E~ (RULE 26) Al~r Blue Assay.
Alamar Blue is a commercial material designed for use in cytotoxicity assays ~or cells in culture. The reagent is reduced intracellularly in an energy dependent ~ashion. The reduced form of the reagent is readily quanti~ied by either its absorption spectrum or by fluorescence. We have quantified the reduced form of Alamar Blue by absorption spectroscopy.

Target n~UNA
The target site in the CAT mRNA corresponds to CAT site 3 described in Haseloff and Gerlach (1988). The cleavage triplet is a GUC site and is located towards the 3' end 662 nucleotides from the ATG start codon.

Se~uences o~ molecules The test molecules are as ~ollows (Seq ID No. 21-24):
Lower case letters are DNA, upper case letters are RNA.

tRNA Yeast tRNA (Sigma) Phenol/Chloro~orm extracted.
Nl, 5' nnn nnn nnn nnn nnn nn 3' (n = a, g, c or t) CAT Antisense 5' att ctg ccg aca tgg aa 3' CAT Minizyme 5' att ctg cc CUGAUGA gtttt GAAAC atg gaa 3' CAT Inactive Minizyme 5' att ctg cc CUGAUGA gtttt GAGAC atg gaa 3' Bold G in CAT Inactive Minizyme represents the mutation that inactivates the minizyme.

~U~lllUl~ SHEET ~R~e26) Results.
Table 3.
Data from three T- ~ , ' ' E,~_.

Treatment Replicas CAT Activity ~ SEM AlamarBlue I SEM

F.X~
serumstarved+ 3 9385 1 318 1.08 1 0.06 lip~,r~ ",;"~,, Mini~me 3 3788 1 603 1.19+0.06 Antisense 3 3885 +490 0.975 1 0.05 Inactive ~ni~me 3 7043 l 695 1.085 ~ 0.01 (n=2) N" 3 7254 1 876 0.966 ~ 0.06 F~ .l 2' serumstarved+ 2 10573~455 1.045 ' 0.03 lip--rrl;lh.ll;..~.
tRNA 3 9680+656 0.938 1 0.04 ~niyme 3 4943 l 974 0.953 ~ 0.02 Antisense 3 5073+467 0.883 1 0.01 InactiveMini~yme 3 6641 1 642 0.872 ~ 0.05 Nl~ 3 4260+342 0.857 1 0.05 serum starved + 2 4143 + 269 0.89 + 0.05 rr~,l n~
tRNA 3 5646 1 235 0.781 1 0.02 Mi~y~le 3 2855 1 491 1.031 ~ 0.07 Antisense 3 29781259 0.712 1 0.10 InactiveM~i,y~; 3 59691225 0.778 1 0.04 Nl, 3 2210 + 195 0.767 1 0.04 Table4 Mean~SEM CAT E.~c~wl~ a~ ~g~ofCon~ol.
(Con~ols ~esen~ns~vcd+ ~ .r~,~ ~ea~ ~lls) Tl~ah~ F~l .;.. ~1 F.l_.;.. f2 F~l_.;.. l3 MeanlSEM
Con~ol 100~3.3 100~4.3 100+6.5 100 ~NA - 91.6~6.8 136.3~4.2 114~22 ~G~y c 40.4+15.9 46.8~19.7 68.9~17.2 52~9 An~s~e 41.4~12.6 48.0~9.2 71.9~8.7 54~9 ~ac~ve M~me 75.0~9.9 62.8+9.7 144.1~3.8 94~25 Nl7 773~12.1 40.3~8.0 53 3 1 8.8 57~11 Figure 1 shows the CAT Expression (Mean + SEM) for Di~erent treatments. Each experiment is per~ormed in triplicate. Figure 2 shows the extent of Alamar Blue Reduction (mean + SEM) in the same experiments as in Figure 1. Extent o~ reduction relates to metabolic activity of the cells during the 18 hours post-transfection.

Discus~ion.
The CAT minizyme contained the new linker 5~gtttt. When tested against a short 17-mer synthetic RNA substrate in vitro at 37'C, the minizyme cleaved the substrate with a reasonable rate constant (- 0.5 min~1, t1/2 ~ 1.4 minutes). In all experiments a constant number o~ cells (8 x 104) were seeded and treated identically with the exception o~ the added oligonucleotides.
Based on observed CAT activity, the random 17-mer does no~
appear to be an appropriate control molecule. Accordingly tRNA
was included in two o~ the experiments to provide an alternative control.

The minizyme and the DNA antisense show similar levels o~
suppression (52 + 9~, 54 + 9~, respectively) and both are signi~icantly more ef~ective than the inactivated minizyme (94 +

SU~ lul~:; SHEET (Rule 26) W096/40906 CA 02222793 l997-l2-Ol PCT/AU96/00343 25~, mean results Table 4). Thus both the minizyme and the antisense are showing activity in this cultured cell system.

We have been mindful of the danger of selecting a single protein or mRNA level as a specificity control and have therefore monitored the rate of general metabolism (by Alamar Blue reduction) as a measure of the specificity of the test molecules.
It is interesting to note that the minizyme is apparently less toxic than the antisense in all experiments (Table 3, Figure 2).
Therefore the relative activity of the minizyme may be greater than that of the antisense, since a proportion o~ the apparent activity of the antisense molecule could result from a reduction in cell metabolism compared with minizyme treated cells.

Ex~mple 3 The activities of DNA-armed ribozymes and minizymes against I~terleukin~2 mRNA in vivo.

The molecules tested.

The ~ollowing molecules (2-7) have been synthesized and tested for activity using one or more of the assays described below.
Molecule 2 is a hammerhead ribozyme with deoxyribonucleotides in the arms which hybridize to the substrate. Molecule 3 is a minimized hammerhead ribozyme (minizyme) with a linker of sequence d(GTTTT) replacing stem-loop II of the full-sized ribozyme. Molecule 4 is a minizyme with 5' d(GTTTT) linker, which has been rendered inactive by replacing the conserved A
by a G. Molecule 5 is a DNA antisense control with sequence complementary to the 15-nucleotide target sequence. Molecule 6 is a DNA control with the same base composition as the DNA
antisense, but with scrambled sequence. Molecule 1 is a 15-nucleotide, synthetic substrate with the same sequence as that targeted in tne IL2 m-RNA; it is used to determine cleavage rate constants for the ribozyme and minizymes in vitro. In the sequences below, ribonucleotides are denoted by SUBSlllUl~ SHEET (Rule 26) ~ =

upper-case letters and deoxyribonucleotides by lower-case letters (Seq ID No. 25-31).

l. S' UCCUGUCWGCAWG interleukin-2 target sequence 2. 3' aggaC aacgtaac 5' \A CUG DNA-armed ribozyme ~ A IL2R-DNA
A ,GU
G A
C.G
~ U
U U
U-U

3. 3' a g g a C\ a a c g t a a c 5' ll~ilJi~yllle with dG'll~T linker A CUG\ IL2M
A A
IA GU
tG gA

t-t-t 4. 3' a g g a C a a c g t a a c 5' inactive lllilliGyllle A CUG\ (GAAAC - GAGAC) 3 o G /A IL2M~c~ve A GU

t g t--t-t W096/40906 CA 02222793 l997-l2-ol PCT/AU96/00343 5. 3' aggacagaacgtaac 5' DNA~nticPn.cecontrol 6. 3' gaatcgcagaaagca 5' DNArandom-sequence control 7. 3' a g g a C a a c g t a a c 5' ~ ~y~l~e with dCCTT linker A A
A GC
G A
t c \~J

Preparation of oligonucleotides The oligonucleotides were synthesised on an Applied Biosystems (Foster City, CA) model 391 synthesiser using protected DNA
phosphoramidite monomers and RNA monomers, protected at the 2'-hydroxyl by tert-butyldimethylsilyl groups, ~rom Millipore (Marlborough, MA). For convenience in the syntheses, the 3' nucleotide in all molecules is a deoxyribonucleotide. The methods used ~or the deprotection and gel-puri~ication o~ the oligonucleotides were as described previously (McCall et al., 1992), with the exception that the oligonucleotides used ~or testing in cells were precipitated twice (rather than once) ~rom 0.3 M sodium acetate and 80~ ethanol, and then washed twice (rather than once) in cold 80~ ethanol be~ore drying under vacuum. The oligonucleotides were re-dissolved in autoclaved, de-ionized water. The concentrations o~ the oligonucleotide solutions were determined by W spectroscopy using the ~ollowing molar extinction coe~ficients ~or the W O~C/109~ CA 02222793 l997-l2-Ol PCT/AU96/00343 nucleotides at 260 nm: A 15,400, G 11,700, C 7,300, T and U 8, 800 Lmol~lcm~'. The purity of each oligonucleotide was checked by phosphorylating the 5' end using [y-3-] ATP and polynucleotide kinase (Boehringer Mannheim, Germany) as described previously (McCall et al., 1992). The oligonucleotides were stored at -20 C.

Dete-~;n;~ rates o~ cleavage (k~,) by minizymes The rates at which the minizymes (IL2M~s~ and IL2~ ~) and ribozyme (IL2R-DNA) cleaved their cognate, short substrate (IL2S15) were determined in 10 mM MgCl, 50 mM Tris.HCl buffer, pH 8.25, at 37'C with 5 ,uM minizyme and 2 ,uM substrate. The substrate was labelled on its 5' end with [32p] -phosphate. The minizyme and substrate were heated together in buffer for two minutes at 70 C, put to 37 C for 2 minutes, centrifuged briefly at room temperature, and then returned to 37 C ~or 2 minutes be~ore starting the reaction by adding MgCl2 (at 37 C) to the mixture. The volume of the reaction mixture was generally 30 ~L. 2 ~L samples were taken at various times, and the reactions in these were quenched by adding 4 ~L of 20 mM EDTA, 80~ formamide, 0.1~ bromphenol blue and 0.1~ xylene cyanol. The samples were electrophoresed on a 15~ polyacrylamide gel containing 7M urea, and the amounts of 5'-product and uncleaved substrate were quanti~ied using a Molecular Dynamics PhosphorImager (Sunnyvale, CA) and ImageQuant software. Kinetic parameters were obtained by fitting the data ~or ~ of product formed (P) versus time (t! to the equation P. = P - (exp(-k~ t)P~) where P is the amount of Froduct at time t, P,is the amount of product at t = a~ kob, is the first-order rate-constant ~or the reaction, and P~ is the difference between the percentage o~
product at t = ~ and t = 0. This is a conventional first-order kinetic equation from which k~, P and P~ are determined by least-squares fitting of the data. The quoted rate constants SUBSmUTE S~EE~ (RULE 26) -W O 96/40906 F'CT/AU9G~

are the mean (+ standard deviation) of at least two experiments.

Assays ior interleukin-2.

Abbreviations: PBMN (peripheral blood mononuclear cells), PHA
(phytohemagglutinin), IL-2 (interleukin-2).

Interleukin-2 is synthesised and secreted by T-lymphocytes following their activation by antigens or mitogens. In experiments to determine how well the test molecules could suppress the production of IL-2, an enriched population o~
human PBMN T-cells were transfected with the test molecules, and then stimulated with PHA to produce IL-2. In all cases, these test molecules were co-transfected with 1 ~g tRNA and their effects on IL-2 levels were determined relative to the effect of the control which was l~g tRNA. The amounts of IL-2 secreted were assayed by three methods. In one method, IL-2 was assayed indirectly by measuring the ability of the supernatant from the PBMN cells to promote the growth o~ IL2-dependent mouse CTLL cells. In another, since IL-2 produced by PBMN
T-cells promotes the growth o~ the same cells, IL-2 levels were assayed directly by measuring the growth o~ the stimulated cells. In addition to these bio-assays, immunoassays (which measure free and receptor-bound IL-2) were performed.

Indirect bio-assay.
This assay is speci~ic for interleukin-2 and is based on the absolute requirement of IL-2 ~or the growth o~ CTLL cells.
Before use, the CTLL cells, maintained continuously proliferating in complete media containing added IL-2, were washed 3 times in complete media without added IL-2(Tables 13a and 13b).

SUBSTITUTE S~tE T tRULE 26) W096/40906 CA 02222793 l997-l2-0l PCT/AU96/00343 In microtitre trays 100,000 PBMN cells (enriched in T-cells) were trans~ected in complete medium (RPMI plus 10~ foetal-cal~
serum) with the test and control molecules at 5, 10 and 20 ~M
using 25 ~g/mL DOTAP (Boehringer Mannheim, Germany) according to the manufacturer's instructions. After a period o~ 7 hours, the cells were stimulated with 5 ~g/mL PHA (Sigma). A~ter an additional 15-20 hours, supernatants ~rom the PBMN cells were serially diluted and, in triplicate, 100 ~L from each dilution were added to 5000 CTLL cells in 20 ~L media. The CTLL cells were allowed to grow ~or 20 hours, were pulsed with 3H-thymidine ~or 4 hours, were harvested, and then the DNA-associated radioactivity was determined. Slow growth of the CTLL cells (low level o~ incorporated 3H-thymidine) indicates low levels o~ interleukin-2 in the supernatant, and hence a test molecule with good activity against interleukin-2 in the PBMN cells. The percentage inhibition of IL-2 by the test molecules is measured in the range where the cell growth-rate is linearly dependent on the amount of IL-2 (Sioud, 1994).

Direct bio-assay.

PBMN cells were transfected with the test molecules ~or 12 hours, stimulated with PHA for about 48 hours, pulsed with 'H-thymidine, and then harvested 18 hours later to determine DNA-associated radioactivity (Tables 14a and 14b).

Enzyme Ampli~ied Sensitivity Tmm~no-As~ay (EASIA).

Total I~-2 in the supernatant ~rom PBMN cells trans~ected with 20~M IL2M~ and 20~M IL2M ,~. was determined using IL2-EASIA, a solid-phase Enzyme Ampli~ied Sensitivity Immuno-Assay per~ormed in microtitre plates according to the manu~acturer~s instructions (MADGENIX) (Table 15).

Toxicity to cells.

SUBSTITUTE S~ EET ~RULE 26) WO~6/1C9~6 CA 02222793 l997-l2-ol PCT/AU96/00343 The viability of PBMN cells which had been transfected with test and control molecules was determined using acridine orange and ethidium bromide. Dead cells appear orange and live cells appear green in the presence of these indicators. The assays were performed on PBMN cells that were transfected with the test and control molecules ~or a period o~ 22-27 hours; these cells were not stimulated with PHA since this results in cell aggregation and hence inaccuracy in cell counting. In parallel, cells ~rom the same donor were transfected with the test and control molecules and stimulated with PHA, and when supernatants ~rom these cells were collected for assaying IL-2 levels, the viabilities of the cells not stimulated by PHA were determined.

Acti~ities of DNA-armed ribG~y -~ A~d minizymes.

The original set of molecules synthesized for testing contained the DNA-armed ribozyme, the minizyme with d(CCTT) linker, the DNA antisense, and the DNA random-sequence control (molecules 2, 7, S and 6 respectively). In these first experiments using the indirect assay, with transfection concentrations of test molecules at lO~M, the DNA-armed ribozyme showed good activity against IL2, while the minizyme with d(CCTT) linker and the DNA
random-sequence control showed no activity; the DNA antisense showed about 50~ the activity of the DNA-armed ribozyme. The activities of the DNA-armed ribozyme and the minizyme in cells correlated with the observed cleavage activities of these molecules as measured against the short synthetic IL2 substrate in vitro. Since the cleavage rate shown by the minizyme with d(CCTT) linker was extremely slow, we investigated the reason for this poor activity. In this investigation we found that by changing the linker from 5' d(CCTT) to 5' d(GTTTT) the cleavage rate o~ the minizyme could be increased 25-fold. The observed cleavage rate constants in lO mM MgCl, at pH 8.25 and 37 C, for the DNA-armed ribozyme, the minizyme with d(CCTT) linker, and the new minizyme with d(GTTTT) lirker, against their SUB5TITUTE SHEET ~RULE 26) cognate, short substrate are 1 4 (0.2), 0.011 (0.001) and 0.27 (0.04) min~l, respectively.

Following the discovery of the highly active minizyme with d(GTTTT) linker, we synthesized in large scale the DNA-armed ribozyme, the minizyme with d(GTTTT) linker, an inactivated minizyme with d(GTTTT) linker, the DNA antisense, and the DNA
random-sequence control (molecules 2-6 above), and tested them for activity in cells Results from inpedendent experiments using molecules 2-6.

In the tables below, the e~fectiveness of each test molecule (at different concentrations) in inhibiting the production of IL2 is presented as ~ inhibition averaged over two or three independent experiments (each done in duplicate or triplicate) with st~n~d deviations given in brackets. In the first of each pair of tables, the data show the effectiveness of the test molecules against IL2, relative to that o~ the DNA
random-sequence control taken as having o~ inhibitory effect Since ribozymes and minizymes are being developed as alternatives to other oligonucleotide-based therapies, we believe that the inhibitory effects of these molecules, over and above the non-specific effects of a randomly-chosen oligonucleotide, are the data of interest. In most experiments, tRNA was also included as an additional control molecule. Generally, the 3NA random-sequence control showed some activity against IL2 relative to tRNA, and so these da.a are presented ~or information (relative to tRNA ta~en as having 0~ inhibitory e~ect) in the second of each pair of tables.

SUBS 111 UTE St~EET (RULE 26) Indirect assay. % inhibition of IL2 as m~a ,..r~d by the indirect assay.
Table 13A. Data relative to DNA random-sequence control.
T~ re-;lion conc~llLl~Lion Test molecule S ~M lO~M 20,uM

l:L2R-DNA 33 42 (13) 69 (1) IL2M,~ 31 41 (4) 53 (11) IL2MI,,~, 5 6 (27) 28 (6) 10 IL2AS 10 21 (19) 13 (6) Table 13B. Data relative to tRNA.

lS Tr~n~fectiQn con~ntration Test molecule 5 ,~M lO,L~M 20~M

IL2R-DNA 45 51 (9) 80 (4) IL2M,~ 43 51 (3) 73 (12) IL2M""~"~ 22 22 (17) 47 (12) IL2AS 25 35 (9) 49 (15) IL2C 18 16 (7) 35 (9) Direct assay. % inhibition of IL2 as mea~,..r~d in individual ~ e. il..ents by the direct assay.
Table 14A. Data relative to DNA random-sequence control.
Transfection conce,lL,alion Test molecule 10,uM

IL2R-DNA 67 (3) IL2M,~ 46 (6) TT ~M. 24 (2) 10 IL2A~ 28 (14) Table 14B. Data relative to tRNA.
Tl~ r~cl;~ n COl~C~llLl~Lion Test molecule 10 M
IL2R-DNA 73 (4) lL2M~
IL2M~ 37 (2) IL2AS 40 (14) IL2C 18 (4) E~SL4 Table 15. T,~ cl;on concenL,~Lion 20,L~M.
Test m~-lecllle IU/ml % inhibition relative to tRNA
IL2M~ 70 72 %
IL2M"~,~" 120 52 %
control (tRNA) 250 WO ~6/109~~ CA 02222793 l997-l2-Ol PCT/AU96/00343 Sl ~~y of results.

The DNA-armed ribozyme (IL2R-DNA) and the minizyme with d(GTTTT) linker (IL2M~) show activity specifically against interleukin-2 in human PBMN cells. The effects of these two molecules are greater than those measured for a DNA antisense molecule (IL2AS) and an inactivated minizyme (IL2M~cr~ve) directed to the same target site on the IL2 mRNA, at transfection concentrations of 5, 10 and 20 ~M. The molecules do not appear to be toxic to cells over a 24-hour period.

Example 4 Mini-ribozymes Comments in publications on h~mmerhead ribozymes teach that helix II can be reduced to 2 b.p. without loss of activity, but further reduction to 1 b.p. results in at least a 10-fold reduction in activity (see data from Tuschi and Eckstein (1993) and from Long and Uhlenbeck (1994)). Note that these data are for all-RNA rlbozymes We have found that ribozymes with 1 b.p. in helix II, such that the sequence of "stem-loop II" is 5'GTTTC or 5'GTTTC, where T may be dT or rU, have better than 10~ the activity of analogous ribozymes with 4 b.p. in helix II. We call such ribozymes "mini-ribozymes". In some cases, the mini-ribozymes have observed cleavage constants which exceed that of the full-sized analogous ribozymes. Based upon published data, these findings are totally unexpected. Our data are presented below.
Seguences of mini-ribozymes and ribozymes.

- Upper-case letters represent ribonucleotides, and lower-case letters represent deoxyribonucleotides. Substrates are labelled S, followed by the number of nucleotides in the substrate molecule. Minizymes are labelled M, followed by the sequence of the linker connecting the conserved nucleotides A~

SU~S~I I UTE SHEET (RULE 26) CA 02222793 l997-l2-Ol and G12 (e.g. KrMgtttc is a miniribozyme in the Kruppel system, with a deoxyribonucleotide linker of sequence 5'd(GTTTTC).
Full-sized ribozymes which contain a stem-loop II (with 1 b.p.
in helix II) are labeled RA (i~ made of ribonucleotides), RB
(with deoxyribonuclotides in the arms which hybridize to the substrates, and ribonucleotides elsewhere), and RC (i~ made of deoxyribonuclotides, except for the conserved r nucleotides C3-Ag and C1z C15.2) Interleukin-2 system (Seq ID No. 32-38) 10IL2S15 ( 15 mer substrate) 5~ UCCUGUCUUGCAW g 3' IL2Mgtttc (DNA/RNA mini-ribozyme with d(GTTTC) linker 3' a-g-g-a-C a7a-c-g-t-a-a-c 5' A CUG\
A A
A GU /
20G\ A

c \ t /

IL2Mgttttc (DNA/RNA mini-ribozyme with d(GTTTTC) linker 3' a-g-g-a-C a-a-c-g-t-a-a-c 5' A CUG
A A

G A

t/
t-t SUBSmUTE S~EET (RULE 26) W096/40906 CA 02222793 l997-l2-ol PCT/AU96/00343 IL2"RB" DNA-armed ribozyme 3' a-g-g-a-C a-a-c-g-t-a-a-c 5' A CUG
A A
A GU /
b Al C.G /
A.IU
G C

U U
U--U/

IL2"RC" DNA-containing ribozyme 3' a-g-g-a-C a-a-c-g-t-a-a-c 5' A CUG\
A A
A GU /
G A
\c.g /
a.t c.c g.c.\
t t \t- t IL2 MGUUUUC(all-RNA miniribozyme with r~GUUUUC) linkers 3' A-G-G-A-C A-A-C-G-T-A-A-C 5' 1 l A CUG
A A
A GU /
G A
\C.G-~
U U
U--U

CA 02222793 l997-l2-Ol IL2 (all RNA ribozyme) 3' A-G-G-A-C A-A-C-G-T-A-A-C 5' A CUG

A GU
G A

\A $
ci . c ~. C \
U U
\ U--U
TAT system (Seq ID No. 39-43) TATS13 (13 mer substrate) 5' GGAAGUCAGCCU a 3' TATS21 (21 mer substrate) 5' UCCAGGA~GUCAGCCUAGGA c3' TATMgtttc (DNA/RNA mini-ribozyme with d(GTTTC) linker 3' aggtcctt-C tcggatcctg 5' A CUG
A
A GU
G A
c g t t \t /

CA 02222793 l997-l2-Ol W O 96/1~3_~ PCT/AU96/00343 TATMgttttc (DNA/RNA mini-ribozyme with d(GTTTTC) linker 3' aggtcctt-C tlcggatcctg 5' A CUG\
A A
A GU /
G A

/c \t-t /

TAT~RC" DNA-containing ribozyme 3' aggtcctt-C\ tcggatcctg 5' AAf CUG\A

GU /
G A/
~c.g /
l,l g . c g.c\

\t_ t/

Kr system (Seq ID No. 44-47) KrS13 (13 mer substrate) - 5' GCGAGUCCACAC T 3' KrS21 (21 mer substrate) W096/40906 CA 02222793 l997-l2-ol PCT/AU96/00343 5' AUUUGCGAGUCCACACACUGGA g 3' KrMgttttc (DNA/RNA mini-ribozyme with d(GTTTTC) linker 3' taaacgct-C gtgtgacctc S' ~A CUG\
A A
A GU /

\c g \

\t-t /

Kr "RC" DNA-containing ribozyme 3' taaacgct-C\ gtgtgacctc 5' A/ CUG~
A
A GU
A

a t . g.c g.c\
t-~ t \t -t Experimental data.

The method ~or determining rates o~ cleavage (kobs) of short substrates by the miniribozymes is as described in Example 1.

W096/40906 CA 02222793 l997-l2-0l PCT/AU96/00343 Initial experiments at pH 8.2 showed the reactions ~or the TAT
and IL2 miniri~ozymes and "RC" ribozymes are very fast, with the reactions being completed in less than 1 minute.
Therefore, the reactions were per~ormed at the lower pH of 7.1 (which in principle should be 12.6-fold slower than the pH 8.2 if the reactions have a first order dependence on OH
concentration in this pH range). The data at pH 7.1 show more accurately the relative activities of the various molecules.
In the Tables below, values in parentheses(a) are the standard deviations of at least two independent determinations; and indicates an initial ~ast reaction followed by a slower reaction, with for the initial fast reaction being given.

Table 8. kd~ (min-1) and ~P~ for KrMgttttc and KrRC cleaving the 13-mer and 21-mer substrates in 10 mM MgCl2 , 50 mM Tris.Cl pH 7.1, 37~C, 6~M ribozyme/miniribozyme and 4~M substrate.

K~M ~ c K~RC

kobs(~ ~POa kObS(a) %P~

K~Sl3 0~9*(009) 70(3) 1.6*(03) 78(2) K~S21 30* (02) 58(1) 134*(008) 56(1) Table 9. kob5 (min~1) and ~P~ for TATMgttte, TATMgtttte and TATRC cleaving various substrates in 10mM MgCl2, 50 mM Tris.Ci pH 7.1, 37~C. Concentration o~ ribozyme exceeded that o~
~ substrate, with concentrations varying between 4~M and 8~M
(ribozyme and miniribozyme) and 2~M and 5~M (substrate), wit;~
typical concentrations being 6~M for ribozyme/miniribozyme and 4~M ~or substrate.

SVBSmVTE S~EET (RULE 26) W O~C/10~06 PCT/AU96/00343 TATMgmc TATM~LtLlc TATRC

ko~ (~) ~pO~ko~ (~) ~POa kobs (~) %POa TATS13 0.06(0.02) 78(6) 0.175* (0.003) 74(7) 0.43* (0.008~ 80 (7) TATS21 1.1*(0.3) 39 (1) 0.9* ~0.2) 45 (3) 0.45* (0.09) 31 (2) Table 10 . kob,; (min~1) and ~PO for the DNA-containing IL2Mgtttt, IL2Mgttttc, IL2Mgtttc, II2RB and IL2RC, and the all-RNA
IL2MGUUUUC and IL2RA cleaving the substrate IL2S15-6/8 in 10 mM
MgCl2, 50 mM Tris. Cl pH 7.1, 37~C. Concentration of ribozyme exceeded that of substrate, with concentrations varying between 2.5 ~M and 20 ~M (ribozyme), and 1/~M and 8~M (substrate, with typical concentrations being 5 ~M for ribozyme/miniribozyme and 2 ~M for substrate.

ko~(a) ~POa IL2Mgtte 0.119 (0.0004) 85(4) IL2Mgtme 0.19 (0.02) 82(1) IL2RB 0.06 (0.02) 78(5) IL2RC 0.05 (0.001) 90(7) 25 IL2MGUUUUC 0.316 (0.0001) 76(2) IL2RA 1.4* (0.2) 66(1) Table 11 . Comparing the rates of cleavage of substrates (at 37~C, pH 7.1, 10 mM MgCl2) by the miniribozymes with d~GTTTC) and d(GTTTTC) linkers relative to that analogous DNA-containing ribozymes RC, and the all-RNA mini-ribozyme with r(GUUUUC) linker relative to the analogous all RNA ribozyme RA.

SUBSTtTUTE SHEET (RULE 26) W O 96/~O~D~ PCT/AU96/00343 kobS gtttc/RC k~ gttttc/RC ko~ GUlnJUC~RA

r K~S13 _ 037 K~S21 - 22 5 TATS13 0.14 0.41 rL2Sl5 2.4 38 0.23 C~ -~ts.
Several of the molecules used (Kr"RC", TAT"RC", IL2S15, IL2Mgttte and IL2"RC") have been synthesized at least twice.
The rates of cleavage and extents of cleavage do not vary significantly for molecules ~rom different syntheses.
The IL2 minizyme with d(GTTTT) linker, which has kobs = 0.27 min~1 at pH 8.2 (and kobs = 0.024 (0.005) min1 at pH 7.1), is active in cells (see Example 3). Therefore, the IL2 mini-ribozymes with d(GTTTC) and d(GTTTTC) linkers, which have kobs = 0.11 and 0.19 min~1, respectively, at pH 7.1, and which target the same site in the IL2 mRNA, are expected to be active in cells. For similar reasons, the all-RNA IL2 mini-ribozyme IL2M~uuuuC, which has kobs = 0.316 min at pH 7.1, is expected to be active in cells.
Example 5 In preliminary experiments, a miniribozyme targeted against interleukin-2 mRNA was shown to have activity against this target in human peripheral blood mononuclear cells. The sequence o~ the miniribozyme, IL2MGU W UC, was 5' CAAUGCAA
CUGAUGA GUUUUC GAAAC AGGa 3' (SEQ. ID NO. 48) where upper-case letters represent RNA and lower-case letters DNA (the 3' nucleotide was DNA ~or convenience in the chemical synthesis).
The experiment to test ~or activity in cells was per~ormed in the manner described in Example 3 (pages 47-57).

SUBSTITUTE SHEET (RULF 26'~

Example 6 The interleukin-2 miniribozyme cleaves an 809-nucleotide interleukin-2 transcript much faster in vitro at 37~C than does an interleukin-2 ribozyme; time taken for 50~ of the IL2 transcript to be cleaved is 30 minutes for the miniribozyme and 4.6 hours for the ribozyme. Also, the miniribozyme cleaves the interleukin-2 transcript faster than does the ribozyme over a wide temperature range (please see Figure 5).

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into conserved nucleotides A6, G8, A9, G12, A13 and Al~
reduces activity, but doesn't eliminate it. Making G5 as DNA results in big reduction in activity.
Perreault, J.-P., Labuda, D., Usman, N., Yang, J.-H. and Cedergren, R. (1991), Biochemistry, 30, 4020-4025. G5 and A9 cannot be DNA. G12, A13, A14, G8 can be DNA.

S~ lul~ S}~:~;T (Rule 20 Perriman, R., et al. (1993) Antisense Res. & Dev. 3:253-263.
Piccirilli, J.A., et al. (1992) Science 256:1420.
Pieken, W.A., Olsen, D.B., Aurup, H., Williams, D.M., Heidenreich, O., Benseler, F., and Eckstein, F. (1991), Nucleic Acids Res. Symposium Series, 24. (2'amino group at C15.2 (together with 3 other 2'-amino Cs in hybridizing arms) has reduction in activity. Can't have 2'-amino at G5, but can at G12.) Pieken, W.A., Olsen, D.B., Benseler, F., Aurup, H. and Eckstein, F. (1991), Science, 253, 314-317. Can make all cytidines (C3, C15.2) and uridines (U4, U7) 2'-fluoro or 2'-amino and not get much reduction in cleavage activity. A9 can be 2'-~luoro, but A13, A14 and A15 together cannot have 2'-~luoro group.
Pyle, A.M. (1993) Science 261:709-714.
Robertson, D.L., and ~oyce, G.F. (1990) Nature 344:467.
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Shimamoto, K., Terada, R., Izawa, T., and Fujimoto, H.
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Sinha, N.D., Biernat, J, & Koster, H. (1984) Nucl. Acids.
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pp 49-86, F. Eckstein (Ed). Oxford University Press, Oxford.

~Slll~ SHEET ~R~e26) Sproat, B. S., A. I. Lamond, R. G. Garcia, B. Beijer, U.
Pieles, M. Douglas, K. Bohmann, F. M. ~armo, S. Weston and S. O'Loughlin. (199lB). 2'-O-alkyloligoribonucleotides, synthesis and applications in molecular biology. Nucleic Acids Symp Ser. 1991 (24). 59-62.
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SUB~ ulk SHEET (Rule 26) Vasseur, J. J., F. Debart, Y. S. Sanghvi and P. D. Cook.
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CAATGCAACU GACGACCTTG A~ACAGGA 28 (2) INFORMATION FOR SEQ ID NO:10:

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CAATGCAACU GAUGANGA~A CAGGA 25 (2) INFORMATION FOR SEQ ID NO:11:

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ATTCTGCCCU GAUGATTTTG A~ACATGGAA 30 (2) INFORMATION FOR SEQ ID NO:17:

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CA 02222793 l997-l2-0l WO 9~'1C90C PCT/AU9G,!t~?~

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CA 02222793 l997-l2-0l CAATGCAACU GAUGAGTTTT GA~ACAGGA 29 (2) INFORMATION FOR SEQ ID NO:28:

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(2) INFORM~TION FOR SEQ ID NO:30:

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(2) INFORMATION FOR SEQ ID NO:33:

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CAATGCAACU GAUGAGTTTC GA~ACAGGA 29 (2) INFORM~TION FOR SEQ ID NO:34:

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CAATGCAACU GAUGAGTTTT CGA~ACAGGA 30 (2) INFORMATION FOR SEQ ID NO:35:

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(2) INFORMATION FOR SEQ ID NO:36:

(i) SEQUENCE CHARACTERISTICS:
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(2) INFORMATION FOR SEQ ID NO:37:

(i) SEQUENCE CHARACTERISTICS:
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CAAUGCAACU GAUGAGUUUU CGA~ACAGGA 30 (2) INFORMATION FOR SEQ ID NO:38:

(i) SEQUENCE CHARACTERISTICS:
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~2) INFORMATION FOR SEQ ID NO:39:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOT-ECULE TYPE: RNA (.genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:

~ --90--(2) INFORMATION FOR SEQ ID NO:40:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: RNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:

(2) INFORMATION FOR SEQ ID NO:41:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:

GTCCTAGGCT CUGAUGAGTT TCGA~ACTTC CTGGA 35 (2) INFORMATION FOR SEQ ID NO:42:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs ~B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NC:42:

CA 02222793 l997-l2-0l W O9G/409~ PCT/AU96/00343 GTCCTAGGCT CUGAUGAGTT TTCGA~ACTT CCTGGA 36 (2) INFORMATION FOR SEQ ID NO:43:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:

(2) INFORM~TION FOR SEQ ID NO:44:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single - (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:

(2) INFORMATION FOR SEQ ID NO:4S:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: RNA ~genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:

(2) INFORM~TION FOR SEQ ID No:46:

~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:

CTCCAGTGTG CUGAUGAGTT TTCGA~ACTC GCA~AT 36 (2) INFORMATION FOR SEQ ID No:47:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:

CTCCAGTGTG CUGAUGAGTC CTTTTGGACG A~ACTCGCAA AT 42 (2) INFORMATION FOR SEQ ID NO:48:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:

CAAUGCAACU GAUGAGUUUU CGA~ACAGGa 30 (2) INFORMATION FOR SEQ ID NO:49:

(i) SEQUENCE CHAR~CTERISTICS:
(A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:

CAAUGCAACU GAUGAGUCCU UUUGGACGA~ ACAGGa 35 (2) INFORMATION FOR SEQ ID NO:50:

(i) SEQUENCE CHAR~CTERISTICS:
(A) LENGTH: 51 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (x ) SEQUENCE DESCRIPTION: SEQ ID NO:50:

Claims (37)

WHAT IS CLAIMED IS:

1. A compound having the formula (Seq ID No. 3):

wherein each X and Q represents a nucleotide which may be the same or different and may be substituted or modified in its sugar, base or phosphate and Q
represents guanosine;

wherein each of (X)n and (X)n' represents an oligonucleotide having a predetermined sequence which is capable of hybridizing with an RNA target sequence to be cleaved;

wherein each of n and n' represents an integer which defines the number of nucleotides in the oligonucleotide;

wherein each solid line represents a chemical linkage providing covalent bonds between the nucleotides located on either side thereof;

wherein a represents an integer which defines a number of nucleotides with the proviso that a may be 0 or 1 and if 0, the A located 5' of (X)a is bonded to the G
located 3' of (X)a; and wherein (T/U)b represents an oligonucleotide with the proviso that b represents an integer which is 3 or 4.

2. The compound of claim 1, wherein the oligonucleotide 3'-(X)n-1-A is 3'-(X)n-2-C-A-.

3. The compound of claim 1, wherein (X)a is absent.

4. The compound of claim 1, wherein the integer b of (T/U)b is equal to 3.

5. The compound of claim 1, wherein the integer b of (T/U)b is equal to 4.

6. The compound of claim 1, wherein each X and Q is a deoxyribonucleotide.

7. The compound of claim 1, wherein each X is a ribonucleotide.

8. The compound of claim 1, wherein (T/U)b is a (T)b.

9. The compound of claim 1, wherein (T/U)b is (U)b.

10. A compound having the formula (Seq ID No. 4):

wherein each X and Q represents a nucleotide which may be the same or different and may be substituted or modified in its sugar, base or phosphate and Q
represents guanosine;

wherein each of (X)n and (X)n' represents an oligonucleotide having a predetermined sequence which is capable of hybridizing with an RNA target sequence to be cleaved;

wherein each of n and n' represents an integer which defines the number of nucleotides in the oligonucleotide;

wherein * represents a base pair between the nucleotides located on either side thereof;

wherein each solid line represents a chemical linkage providing covalent bonds between the nucleotides located on either side thereof;

wherein a represents an integer which defines a number of nucleotides with the proviso that a may be 0 or 1 and if 0, the A located 5' of (X)a is bonded to the G
located 3' of (X)a;

wherein (T/U)b represents an oligonucleotide with the proviso that b represents an integer which is 3 or 4.

11. The compound of claim 10, wherein the oligonucleotide 3'-(X)n- is 3'-(X)n-1-A-.

12. The compound of claim 10, wherein the oligonucleotide 3'-(X)n- is 3'-(X)n-2-C-A-.

13. The compound of claim 10, wherein (X)a is absent.

14. The compound of claim 10, wherein the integer b of (T/U)b is equal to 3.

15. The compound of claim 10, wherein the integer b of (T/U)b is equal to 4.

16. The compound of claim 10, wherein each X is a deoxyribonucleotide.

17. The compound of claim 10, wherein each X is a ribonucleotide and GUUU(U)C is RNA.

18. The compound of claim 10, wherein (T/U)b is a (T)b.

19. A composition which comprises a compound of claim 1 in association with an acceptable carrier.

20. A composition which comprises a compound of claim 10 in association with an acceptable carrier.

21. A oligonucleotide transfer vector containing a nucleotide sequence which on transcription gives rise to the compound of claim 1 or claim 10.

22. The transfer vector of claim 21, wherein the transfer vector is a bacterial plasmid, a bacteriophage DNA, a cosmid, or an eukaryotic viral DNA.

23. The oligonucleotide transfer vector of claim 21, wherein the oligonucleotide transfer vector is a plant DNA virus, a geminivirus or an infective phage particle.

24. The oligonucleotide transfer vector of claim 21, wherein the oligonucleotide transfer vector is packaged and contains the promoter sequences for RNA polymerase II or RNA polymerase III.

25. A host cell transformed by the transfer vector of claim 21.

26. The host cell of claim 25, wherein the host cell is a prokaryotic host cell or an eukaryotic host cell.

27. The prokaryotic host cell of claim 26, wherein the prokaryotic host cell is an E. coli host cell.

28. The eukaryotic host cell of claim 26, wherein the eukaryotic host cell is a monkey COS host cell, a Chinese hamster ovary host cell, a mammalian host cell or a plant host cell.

29. A method of cleaving a target mRNA in a subject which comprises administering to the subject an effective amount of the compound of claim 1 or 10.

30. A method of claim 29, wherein the administration is topical.

31. A method of claim 30, wherein the topically administered amount is between 1 ng and 10 mg.

32. A method of claim 29, wherein the administration is systemic.

33. A method of claim 32, wherein the systemically administered amount is between 1 ng and 500 µg/kg weight/day.

34. A method of claim 29, wherein the administration is by aerosol.

35. A method of cleaving a target mRNA in a host cell which comprises administering to the host cell an effective amount of a compound of claim 1 or 10 or a transfer vector which on transcription expresses the compound.

36. The compound of claim 1 or 10 which further comprises an antisense nucleic acid which is capable of hybridizing with an RNA target sequence.

37. The compound of claim 1 or 10 which further comprises at least one additional non-naturally occurring oligonucleotide compound which comprises nucleotides whose sequence defines a conserved catalytic region and nucleotides whose sequence is capable of hybridizing with a predetermined target sequence. The compound of claim 37 wherein the additional non-naturally occurring oligonucleotide compound is a hammerhead ribozyme, a miniribozyme, a minizyme, a hairpin ribozyme, a hepatitis delta ribozyme, an RNAase P ribozyme or a combination thereof.